The FASEB Journal express article 10.1096/fj.03-1263fje. Published online May 7, 2004.
Toll-like receptor 4 functions intracellularly in human coronary artery endothelial cells: roles of LBP and sCD14 in mediating LPS-responses
Stefan Dunzendorfer, Hyun-Ku Lee, Katrin Soldau, and Peter S. Tobias
The Scripps Research Institute, Department of Immunology, 10550 North Torrey Pines Road, La Jolla, CA 92037
Corresponding author: Peter S. Tobias, The Scripps Research Institute, Department of Immunology IMM-12, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail:
tobias@https://www.doczj.com/doc/6d4580540.html,
ABSTRACT
Endothelial cells are activated by microbial agonists through Toll-like receptors (TLRs) to express inflammatory mediators; this is of significance in acute as well as chronic inflammatory states such as septic shock and atherosclerosis, respectively. We investigated mechanisms of lipopolysaccharide (LPS)-induced cell activation in human coronary artery endothelial cells (HCAEC) using a combination of FACS, confocal microscopy, RT-PCR, and functional assays. We found that TLR4, in contrast to TLR2, is not only located intracellularly but also functions intracellularly. That being the case, internalization of LPS is required for activation. We further characterized the HCAEC LPS uptake system and found that HCAEC exhibit an effective LPS uptake only in the presence of LPS binding protein (LBP). In addition to its function as a catalyst for LPS-CD14 complex formation, LBP enables HCAEC activation at low LPS concentrations by facilitating the uptake, and therefore delivery, of LPS-CD14 complexes to intracellular TLR4-MD-2. LBP-dependent uptake involves a scavenger receptor pathway. Our findings may be of pathophysiological relevance in the initial response of the organism to infection. Results further suggest that LBP levels, which vary as LBP is an acute phase reactant, could be relevant to initiating inflammatory responses in the vasculature in response to chronic or recurring low LPS. Key words: innate immunity ? endothelium ? endotoxin
E
ndothelial cells (EC), which form the inner vascular lining, are important in the regulation of vascular tone, coagulation and fibrinolysis, cellular growth, differentiation, and last but not least, immune and inflammatory responses (1, 2). Moreover, these cells are targets for many endogenous and exogenous agents (e.g., lipopolysaccharide [LPS]), that activate the endothelium and result in production of proinflammatory mediators (3). These mechanisms play roles in Gram-negative sepsis, where the body may be overwhelmed with LPS and also in diseases that are linked to a low, but chronic, LPS burden. There is considerable evidence that endotoxin (LPS) contributes to the propagation of atherosclerosis (4, 5), which shares many features with inflammatory diseases (6).
LPS is a major component of the outer surface of Gram-negative bacteria and a potent activator of cells of the immune and inflammatory system, including endothelial cells. In vivo, LPS-induced cell activation depends on the presence of at least four proteins: TLR4 (7–9), MD-2 (10), CD14 (11), and LPS binding protein (LBP) (12). The first three may be present as a complex on the surfaces of myeloid (and transfected) cells (13). Many cells, among them epithelial and endothelial cells, do not express the membrane bound form of CD14 (mCD14); their activation involves soluble CD14 (sCD14) (14). LPS·sCD14 complexes will form slowly in vitro (15), but, in vivo, plasma LBP (16–18) catalyzes LPS·CD14 complex formation (19). LPS·CD14 complexes, whether membrane bound or soluble, are necessary for LPS binding to TLR4-MD-2 (20). In mCD14 expressing cells, most LPS appears to be internalized quite independently of activation (21), and mCD14-negative cells also internalize LPS (22, 23). Although LPS internalization by phagocytes has generally been regarded as a disposal function (21), studies have also suggested that internalization might be required for LPS signaling in myeloid lineage cells (24, 25). However, a very recent publication clearly demonstrates that signaling starts on the surface of TLR4-transfected and TLR4 surface expressing cells when incubated with LPS-sCD14 complexes, even in the absence of LPS uptake (26). Other recent work suggests that internalization of LPS is required for activation of murine intestinal epithelial cells, where mCD14, but not TLR4, is found on the surface (27, 28).
MATERIALS AND METHODS
Antibodies
Anti-TLR4 polyclonal antibody (pAb) H80 and monoclonal antibody (mAb) HTA125 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-TLR2 2392 was a gift of Paul Godowski (Genentech), and anti-TLR4 HTA1216, HTA414, and HTA405 were gifts from Kensuke Miyake (Saga Medical School, Japan). Anti-LBP mAb 2B5 and anti-CD14 mAb 28C5 were produced in our laboratory (29). Polyclonal rabbit anti-human MyD88 was from Imgenex (San Diego, CA). The monoclonal mouse anti-human CD14 (clone M5E2), mouse anti-human CD144 (clone 55-7H1), mouse anti-human CD106 (VCAM-1; clone 51-10C9), rat anti-mouse CD31 (PECAM-1) (clone MEC13.3), rat anti-mouse CD144 (VE-cadherin; clone 11D4.1), and rat anti-mouse CD106 (VCAM-1; clone 429-MVCAM.A) used for immunofluorescence staining and PE-conjugated anti-human CD62E (E-selectin/ELAM; clone 68-5H11) were from PharMingen (San Diego, CA). All secondary antibodies used in fluorescence microscopy were Alexa Fluor conjugates (Molecular Probes, Eugene, OR). PE-conjugated secondary antibodies used in flow cytometry were purchased from PharMingen (San Diego, CA).
Proteins, LPS, and other reagents
Human recombinant sCD14 and LBP were expressed in and purified from BTI-TN-5B1-4 cells (Invitrogen, San Diego, CA) as recently described (23). Both proteins can be radiolabeled at an artificially introduced protein kinase A site. Macrophage-activating lipopeptide-2 (MALP-2) was from Alexis Biochemicals (San Diego, CA), recombinant mouse TNF-α was purchased from Serotec (Oxford, UK), and hIL-1β was from Sigma (St. Louis, MO). Compound 406 was generously provided by Professor S. Kusumoto (Osaka University, Japan). All LPS was purchased from List Biological Laboratories (Campbell, CA). Escherichia coli (O55:B5) LPS
was exclusively used for cell stimulation and tritium-labeled LPS (3H-LPS) from E. coli (K12 LCD25) was used in LPS uptake experiments. For use in fluorescence microscopy, LPS from Salmonella minnesota (Re595) was labeled with FITC (Molecular Probes) (19). Complexes of sCD14 with either LPS were preformed without LBP as described previously (30). Under these conditions, no LPS remains unbound. Complexed LPS (LPS·CD14, FITC-LPS·CD14, 3H-LPS·CD14) was prepared freshly each time and used right away.
Cell culture
HCAEC and human umbilical vein endothelial cells (HUVEC) were purchased from Clonetics (San Diego, CA) and grown to confluence in full growth medium (EGM-2-MV) from the same company. HCAEC were from healthy young donors and were used for experiments from passages 4–8. There was no obvious difference in TLR2 surface expression or responsiveness to various stimuli between passage 4 and passage 8 cells. Medium was changed every day, and cells were passaged in a 1:3 ratio using trypsin/EDTA (0.025%/0.2 mM).
Mouse endothelial cell culture
Mouse microvascular and vascular EC were isolated from lung tissue and the aortas of C57/BL6 mice. Halothane-anesthetized animals were killed, and the lungs and the aorta were explanted under sterile conditions, minced, and digested for 1 h at 37°C in 1 mg/ml collagenase type II (Invitrogen). Cells were stained with rat anti-mouse CD31 (10 μg/ml), and goat anti-rat IgG conjugated to paramagnetic beads (Miltenyi Biotec, Auburn, CA) enabled further purification using MACS (magnetic cell sorting). Endothelial cells were then cultured and maintained on fibronectin-coated tissue culture plates in mouse brain microvascular growth medium (Cell Application Incorporation, San Diego, CA) supplemented with 15 ng/ml basic fibroblast growth factor (Sigma). Purity of the cells was >90% as determined by staining for mouse CD31, mouse CD106, and mouse CD144 and by uptake of Alexa Fluor 488-labeled AcLDL (Molecular Probes).
Monocyte isolation
Human peripheral blood monocytes were purified by MACS (Miltenyi Biotec) according to the manufacturer’s protocol. Purity of the preparations and viability of the cells regularly exceeded 95% as determined by subsequent FACS analyses using FITC-anti-human CD14 and propidium iodide (2.5 μg/ml). The isolation procedure did not activate the cells and did not diminish their ability to be stimulated.
Flow cytometry
For flow cytometry analysis of protein surface expression, HCAEC cultured in six-well plates were harvested with a cell scraper. Freshly prepared monocytes and HCAEC were washed twice and stained with various primary and isotype control antibodies (10 μg/ml) for 30 min at 4°C in PBS/5% FCS containing 0.1% sodium azide. After additional washing steps, cells were incubated with secondary PE-conjugated antibodies for a further 30 min at 4°C, washed and resuspended in PBS, and analyzed on a FACScan (BD Biosciences, San Diego, CA). HCAEC activation was determined by CD62E surface expression, which was quantified using PE-
conjugated mouse anti-human CD62E or control PE-conjugated mouse IgG for 30 min at 4°C before FACS analyses.
RT-PCR
RNA was purified from LPS-stimulated or unstimulated HCAEC or monocytes using the Absolutely RNA RT-PCR Miniprep Kit from Stratagene (La Jolla, CA). mRNA levels of TLR2, TLR4, MD-2, CD14, LBP, and gp96 were determined by RT-PCR. Briefly, the first-strand cDNA was reverse-transcribed from 1 μg total RNA with random primers using the SuperScript first-strand Synthesis System from Invitrogen. The cDNA product was amplified by 30 cycles of 30 s at 94°C, 45 s at 55°C, and 2 min extension at 72°C using specific primer pairs. cDNA from monocytes or from stimulated HepG2 cells was used as control for CD14 or LBP, respectively. The RT-PCR products were separated in 1.2% (wt/vol) agarose gels and visualized with ethidium bromide.
Immunofluorescence staining and fluorescence microscopy
HCAEC were cultured in eight-well Nunc Lab-Tec II chambered coverglasses (Nalge Nunc, Naperville, IL) and after an incubation period of 4 h with either LPS (100 ng/ml), FITC-LPS (100 ng/ml), sCD14 (1.6 μg), LBP (2.5 μg), or preformed FITC-LPS·CD14 complexes (100 ng/ml), cells were fixed and permeabilized using the Cytofix/Cytoperm kit from PharMingen (San Diego, CA) according to the manufacturer’s protocol. Nonspecific binding sites were blocked with 0.5% BSA for 30 min at room temperature, and thereafter the following primary antibodies were added at the indicated concentration for 1 h at room temperature: H80 (20 μg/ml), HTA125 (10 μg/ml), 2B5 (10 μg/ml), anti-hCD14 (10 μg/ml), anti-hCD144 (10 μg/ml), anti-hVCAM-1 (10 μg/ml). Fluorescent wheat germ agglutinin (Oregon Green 488; Molecular Probes) was used for Golgi staining, and fluorescent phallotoxin was used for F-actin staining (both at 5 μg/ml). Cells were washed, and secondary fluorescence-labeled antibodies were added at 5 μg/ml for 1 h at room temperature. Nuclei were counterstained with DAPI (300 nM) for 3 min before cells were extensively washed and treated with SlowFade Light antifade reagent (Molecular Probes). Monocytes were stained in suspension. Thereafter, they were mounted to a cover glass with ProLong (Molecular Probes) mounting medium, thus maintaining their round shape. Images were acquired using the DeltaVision Optical Sectioning Microscope (Model 283). Following data acquisition using the Photometrics CH350L liquid-cooled CCD camera, which collects low-intensity signals with a linear response to light intensity, the data are then deconvoluted using DeltaVision software SoftWoRx 2.5 based on the Agard/Sadat inverse matrix algorithm. Following computational deconvolution, this system can provide high-resolution 3D images of cells, but each single optical section can still be viewed alone.
Cell stimulation experiments
HCAEC and HUVEC were cultured in six-well plates, and after reaching confluence, EGM-2-MV was changed to DMEM/2% FCS or DMEM/2% depleted FCS at least 6 h before an experiment was started. Antibodies were added 30 min before cells were stimulated for 4 h at 37°C with LPS, LPS·CD14, or MALP-2 at various concentrations. Thereafter, the cells were harvested and stained for flow cytometry analyses. Human peripheral blood monocytes were resuspended in DMEM/2% FCS and seeded into 24-well tissue culture plates at 1 × 104
cells/well. After addition of antibodies for 30 min, cells were stimulated with either LPS or MALP-2 (1 ng/ml) for 4 h at 37°C. Supernatants were harvested, and hIL-8 was measured with an ELISA (Biosource, Camarillo, CA). Mouse EC were stimulated with LPS, MALP-2, or cytokines for 12 h in culture medium containing 5% FCS. Thereafter, medium was measured for mouse macrophage inflammatory protein-2 (MIP-2), which is the murine homologue of human IL-8, by ELISA (R&D Systems, Minneapolis, MN). For stimulation of HCAEC with low LPS concentrations or with preformed LPS·CD14 complexes, it was necessary to deplete serum of sCD14 or LBP using anti-CD14 (mAb 28C5) and anti-LBP (mAb 2B5) as described previously (21, 23). Depleted FCS contained <1 ng/ml of remaining antibodies (measured by ELISA). Both CD14 and LBP were below the detection limit of Western blotting (1 ng of protein in our experiments).
LPS uptake experiments
HCAEC, or mouse EC, were grown to confluence in six-well tissue culture plates, and medium was changed to DMEM/2% FCS before experiments. 3H-LPS (100 ng/ml) or 3H-LPS·CD14 (100 ng/ml) was incubated for 2 h with the cells of one well along with various antibodies, sCD14, and LBP. Thereafter, the adherent cells were washed twice with PBS and further treated with 250 μg/ml proteinase K (Sigma) in HBSS for 30 min at room temperature to remove cell surface proteins/receptors (22). The 3H counts remaining after proteinase K treatment were therefore considered to be intracellular. This treatment also detached the cells, which were harvested, washed twice in PBS, and lysed in 300 μl 2% SDS/50 mM EDTA. Liquid samples were transferred to scintillation vials and assayed for counts in Ecoscint scintillation fluid (National Diagnostics, Atlanta, GA). The same procedure was carried out in experiments where 32P-labeled LBP (250 ng/ml) uptake was investigated.
Transient transfection
One day after seeding into six-well plates, HCAEC (5×105 cells/well) were transiently transfected with cDNA for CD14 or TLR4 in pFLAG-CMV.1 (Sigma) using 6 μl/well Lipofectin Reagent (Invitrogen) and 1.6 μg/well DNA in 100μl Opti-MEM I (Gibco BRL, Gaithersburg, MD). After 3 h under serum-free conditions, EGM-2-MV was added and cells were analyzed the following day for surface expression of CD14 or TLR4. In several experiments, transfection efficiency was typically 5–10%.
Statistics
Data are expressed as mean and standard error of the mean (SE). Means were compared by Kruskal-Wallis ANOVA and by Mann Whitney U test. A difference with P < 0.05 was considered significant. Statistical analyses were calculated using the StatView software package (Abacus Concepts, Berkeley, CA).
RESULTS
FACS analyses of HCAEC do not detect surface TLR4
To investigate cell surface expression of TLR2 and TLR4 protein, we performed several FACS analyses on monocytes and HCAEC and compared the results with the TLR2 and TLR4 mRNA
expression levels (Fig. 1). Nonpermeabilized HCAEC or monocytes were stained with 5 different anti-TLR4 (monoclonal HTA1216, HTA405, HTA414, HTA125, and polyclonal H80), anti-TLR2 (mAb 2392), or anti-CD14 (M5E2) primary antibodies. Dot blot assays with immobilized primary antibody confirmed that the secondary antibodies used were able to detect their primary partners (not shown). In contrast to human peripheral blood monocytes, which showed clear surface staining for TLR2, TLR4, and CD14, we could not detect TLR4 or CD14 on the surface of HCAEC, although the TLR4 mRNA expression was similar in monocytes and HCAEC. At an expression level of TLR2 mRNA (after induction with LPS-IFNγ or TNF-IFNγ) comparable to basal TLR4 mRNA levels, 92–98% of HCAEC stained positive for surface TLR2; even in unstimulated HCAEC, where almost no TLR2 mRNA was found, FACS still was able to detect surface TLR2. Although LPS-IFNγ enhanced TLR4 mRNA expression in HCAEC, TLR4 was still not detected on the surface. Figure 1 (HCAEC) shows a representative result of many experiments; in this experiment, HTA125 was used for TLR4 and anti-TLR2 2392 for TLR2 detection. Both antibodies worked very well in monocytes (see Fig. 1, Monocytes). However, upon transient transfection and overexpression of TLR4 or CD14 in HCAEC, some surface expression (~1% of cells) was seen in FACS analyses (see Fig. 1, HCAEC transfected), establishing that the detection system works in HCAEC.
HCAEC express RNA for TLR2, TLR4, MD-2, and gp96, but not CD14 or LBP
To assess mRNA expression in HCAEC, RT-PCR analyses was performed. Agarose gel electrophoresis revealed clear bands at the expected fragment sizes for TLR2, TLR4, and MD-2. Stimulation of the HCAEC for 12 h with 10 ng/ml of E. coli LPS (O55:B5) did not significantly change TLR4 or MD-2 mRNA expression, but enhanced TLR2 mRNA. More important, neither stimulated nor unstimulated HCAEC were positive for LBP or CD14. As expected, mRNA for the chaperon protein gp96 was found in three different HCAEC preparations and in monocytes (Fig. 2).
TLR4 is intracellular in HCAEC and on the surface of monocytes
Expression of TLR4 mRNA in the absence of cell surface TLR4 led us to search for intracellular TLR4 in HCAEC by 3D-deconvolution confocal microscopy. In HCAEC, which are nontransfected cells, TLR4 was found exclusively around the nucleus (Fig. 3A–D). After stimulation of the cells, VCAM-1 was found intracellularly and surface expressed (Fig. 3C). Nonimmune rabbit IgG was used as control and revealed uniform background staining (not shown). In monocytes, use of the same anti-TLR4 antibody (H80), resulted in bright surface staining and showed colocalization of TLR4 to membrane CD14 (Fig. 3E).
Anti-TLR4 antibodies do not block HCAEC response to LPS
HCAEC activation was determined by measuring CD62E surface expression (Fig. 4). Preliminary experiments showed that CD62E is minimally expressed on resting cells, but is rapidly induced and highly expressed upon stimulation. HCAEC were incubated with various antibodies 30 min before stimulation with LPS (10 ng/ml) for 4 h. All anti-TLR4 antibodies used failed to inhibit LPS-induced activation of HCAEC. Even long-term preincubation (up to 12 h) of HCAEC with anti-TLR4 was not able to block LPS activation (not shown). This is in contrast to monocytes, where HTA405 or HTA125 significantly, but incompletely, inhibited LPS-
induced IL-8 secretion. The effects of HTA405 on HCAEC or monocyte activation by LPS were
further explored at a wide range of LPS and mAb concentrations (see Table 1). In no case were
the results qualitatively different from Figure 4. As anticipated from published work, the ability
of LPS to stimulate cells was abolished by anti-LBP (mAb 2B5) or anti-CD14 (mAb 28C5) in
endothelial cells as well as in monocytes. Anti-TLR2 (mAb 2392) completely blocked MALP-2-
induced cell activation in both cell types (Fig. 4). The fact that anti-TLR2 did not inhibit LPS-
induced cell activation excludes the possibility that TLR2 agonist impurities in the LPS
preparation were responsible for activation of the cells. Similarly, lack of an effect of 2B5 on
MALP-2-initiated activation suggests that there is not an LPS contaminant in MALP-2. In contrast to anti-TLR4, Compound 406 (lipid 4A) dose-dependently (0.1–100 μg/ml) inhibited
LPS-induced HCAEC activation (not shown).
TLR4 is required for LPS signaling but not for LPS uptake in EC
We cultured pulmonary microvascular and aortic EC derived from TLR4 –/– C57/BL6 mice and
compared their response to LPS and other proinflammatory stimuli with cells from wild-type
animals (Fig. 5). Compared with cultured HCAEC, the mouse cells did not respond well with
CD62E expression to any stimulus, and therefore we measured MIP-2 secreted into the medium.
Even after 12 h incubation with 10 ng/ml of E. c oli LPS (O55:B5), the TLR4 –/– cells were still
completely unresponsive to this stimulus, whereas the response to murine TNF (10 ng/ml),
MALP-2 (100 ng/ml), or human IL-1 (10 ng/ml) was independent of TLR4 genotype. The wild-
type cells were activated by all stimuli. Results shown (Fig. 5A) are from experiments with
murine aortic EC; lung microvascular EC responded similarly (data not shown). When TLR4 –/–
cells were incubated with 3H-LPS (100 ng/ml) for 2 h with various concentrations of LBP and 3H-LPS uptake was measured, no difference was found compared with wild-type cells, suggesting that uptake occurs independently of TLR4 phenotype (Fig. 5B). Moreover, mouse EC
LPS uptake mimicked the LPS uptake observed in human cells (HCAEC).
LBP facilitates the uptake of LPS and LPS·CD14 complexes into HCAEC
Because previous data show that sCD14 and LBP are required for LPS-induced HCAEC
activation, we further investigated the role of these proteins in LPS uptake. HCAEC were
incubated for 2 h with 100 ng/ml of 3H-LPS along with an increasing amount of human LBP
(Fig. 6A). LBP dose-dependently stimulated 3H-LPS uptake into cells. The maximum of the
effect was seen close to equimolarity of LPS with LBP and resulted in a ~10-fold increase in the
amount of LPS transported into the cells. Because LPS·CD14 complexes are necessary for cell
activation, we were curious whether LBP would enhance uptake of complexes preformed
without LBP, and found that LBP did enhance 3H-LPS·CD14 complex uptake ~5-fold. LBP-
enhanced LPS and LPS·CD14 complex uptake was sensitive to an anti-LBP antibody (mAb
2B5), but was never blocked completely and a low basal uptake remained. This suggests that
LBP is not absolutely necessary for the uptake of LPS and LPS·CD14 complexes. This basal
uptake was insensitive to either anti-TLR4 (mAb HTA405), anti-LBP (mAb 2B5), or anti-CD14
(mAb 28C5) (data not shown).
Similar patterns of uptake are seen when LPS, sCD14, and LBP were added simultaneously and
therefore CD14 complexation of LPS was catalyzed by LBP (Fig. 6B). Thus LPS in the absence
of LBP, alone or in the presence of sCD14, is only minimally internalized as compared with LPS
in the presence of LBP. When LBP catalyzed complexation of LPS to sCD14 is inhibited by anti-CD14 (mAb 28C5), uptake follows the LPS-LBP pattern (10-fold increase). When complexation of LPS to either sCD14 or LBP is inhibited by anti-LBP (mAb 2B5), the effects of both LBP and CD14 are abolished. Finally, data obtained with 32P-LBP (Fig. 6C) show that LBP is internalized with LPS and that the uptake follows the same pattern as observed when LPS uptake was measured.
LPS, LBP, and CD14 all colocalize with intracellular TLR4
In fact, LPS complexes are found intracellularly. HCAEC were incubated for 4 h with FITC-LPS (100 ng/ml) alone or with sCD14 (1.6 μg/ml), LBP (2.5 μg/ml), or both. Thereafter, cells were stained for TLR4, LBP, or CD14 (Fig. 7). Confocal microscopy showed clear colocalization of FITC-LPS and LBP (Fig. 7A), FITC-LPS and intracellular TLR4 (Fig. 7B), LBP and TLR4 in the presence of sCD14 (Fig. 7C), and finally sCD14 and TLR4 in the presence of LBP (Fig. 7D). Under serum-free conditions (without sCD14), where no cell activation occurs, no colocalization of either FITC-LPS (Fig. 7E) or LBP (Fig. 7F) with intracellular TLR4 was observed. Colocalization of LBP and CD14 could not be shown because both are detected by mouse antibodies and these could not be distinguished by labeled second antibodies. LBP or CD14 were never detected intracellularly when added without LPS; instead, large aggregates were found in the microscopic field outside the cells. When HCAEC were stimulated for 2 h with LPS (10 ng/ml), the intracellular adaptor molecule MyD88, which is involved in TLR4 signaling, was recruited to a perinuclear region (Fig. 7G). This was not the case in the absence of sCD14 (Fig. 7H).
LBP enhances HCAEC activation at low LPS concentrations via facilitated LPS·CD14 uptake
Because the preceding experiments revealed a role for LBP in LPS uptake, we were curious whether this would have an impact on cell activation. To exclude additional LPS complex formation during the experiments, CD14-depleted FCS was used for experiments with preformed LPS·CD14 complexes and, because we wanted to see effects of added recombinant LBP, LBP-depleted FCS was used for experiments with uncomplexed LPS. Neither CD14 nor LBP at the concentrations used in our experiments activated cells without LPS.
Figure 8A shows that 10 ng/ml of LPS with 250 ng/ml LBP enabled a full response of the cells and sCD14 (160 ng/ml) showed no additional effect. When LPS and sCD14 were added without LBP under these conditions, a minimal stimulation was observed, probably due to incomplete LBP depletion of the serum. Stimulation under each condition was blocked by an anti-LBP antibody, thus confirming the necessity of LBP for LPS·CD14 complex formation during the 4 h course of these experiments.
In contrast, 10 ng/ml of preformed LPS·CD14 complexes stimulated the cells without any effect of anti-LBP or additional LBP (Fig. 8C), suggesting that LBP is not absolutely required (Fig. 6A) for sufficient uptake of preformed LPS·CD14 complexes at 10 ng/ml. However, uptake did occur, because a blocking anti-TLR4 (mAb HTA405), which would block surface TLR4, failed to inhibit activation even when added together with anti-LBP (mAb 2B5).
At a low LPS concentration (0.1 ng/ml) (Fig. 8B), addition of sCD14 (100 ng/ml) did not stimulate cells. Providing LBP (200 ng/ml) enabled a response of the cells, which was further enhanced by additional sCD14. Activation was again blocked by anti-LBP (mAb 2B5), which would also inhibit rapid LBP catalyzed formation of stimulatory LPS·CD14 complexes. Preformed LPS·CD14 complexes at 0.1 ng/ml (Fig. 8D) did not stimulate cells in CD14-depleted FCS. When additional LBP was provided, cell stimulation was observed, which cannot be explained by LBP catalyzed LPS·sCD14 complex formation, because preformed complexes were added in CD14 depleted medium. Again, stimulation was insensitive to anti-TLR4 (mAb HTA405) and therefore mediated by intracellular TLR4.
Thus, at 0.1 ng/ml of LPS, LBP enhances the uptake of LPS·CD14 complexes, preformed in vitro (Fig. 8D) or formed by LBP-mediated catalysis (Fig. 8B), and this LBP-enhanced uptake results in HCAEC activation.
LBP-stimulated LPS uptake into HCAEC involves a scavenger receptor pathway
Some scavenger receptors have been shown to be lipid A or LPS uptake receptors (31–33). Because HCAEC were found to be CD14 negative, we were curious whether scavenger receptor blocking agents would affect the LPS uptake in these cells. HCAEC were pretreated with dextran sulfate (MW 8000), polyinosinic acid (PIA), polyadenylic acid (PAA), or polycytidylic acid (PCA) for 30 min before measurement of 3H-LPS uptake with or without additional LBP. Both dextran and PIA reduced the LBP-enhanced 3H-LPS uptake to the level of basal 3H-LPS uptake, whereas the polycationic substances PAA and PCA lacked this effect (Fig. 9). Unfortunately, both dextran sulfate and PIA activated HCAEC to express surface CD62E even in the absence of added LPS (data not shown).
DISCUSSION
To begin a study of regulation and modification of TLR2 and TLR4 expression in HCAEC under various conditions, we performed FACS analyses on those cells, but despite the use of several different anti-TLR4 antibodies, we never observed positive surface staining for TLR4. Nevertheless, the cells responded well to both TLR2 and TLR4 agonists, and mRNA for both TLR2 and TLR4 was expressed. TLR2 mRNA was only weakly detectable in resting cells, but the TLR2 protein was still found on the HCAEC surface by FACS, confirming the high sensitivity of this method. After stimulation of the cells, TLR2 mRNA levels corresponded to TLR2 surface expression. However, this was not observed for TLR4. Despite high expression of TLR4 mRNA in both monocytes and HCAEC, TLR4 protein was never seen on the surface of HCAEC. The reliability of the detection system was confirmed in monocytes, which stained well for TLR2 and TLR4 on the surface, and in transfected HCAEC, where some TLR4 surface protein could be detected by FACS after transfection and overexpression of human TLR4.
With the knowledge that TLR4 has been shown to reside in the Golgi apparatus in murine intestinal epithelial cells (28) and that LPS is transported to the Golgi in human epithelial HeLa cells (34), we further investigated HCAEC using confocal microscopy and found TLR4 located in a perinuclear region but not on the surface, where VCAM-1 was observed after cell stimulation. This is in contrast to monocytes, where TLR4 was clearly seen to colocalize with
mCD14 on the plasma membrane. Our data confirm in part the finding by Faure et al. (35). They described the up-regulation of TLR2 mRNA and TLR4 mRNA upon LPS and IFNγ stimulation in aortic EC. However, surface expression was only shown for TLR2 and not for TLR4. Because the number of TLR4 molecules on the HCAEC surface may be below the detection limit of either confocal microscopy, FACS technology (which has been proven to be very sensitive in TLR2 detection), or immunohistochemistry, we proceeded with the most sensitive method and performed functional assays.
None of the anti-TLR4 antibodies were able to reduce LPS-induced CD62E (E-selectin) expression in HCAEC, although HTA405 and HTA125 blocked LPS-induced IL-8 production by human peripheral blood monocytes. Given that monocytes express large amounts of TLR4 on the surface, as seen in FACS and microscopy, an equal amount of an anti-TLR4 antibody should have been able to block only a few surface TLR4 molecules on endothelial cells if the TLR4 were there. In contrast to the anti-TLR4 antibodies, comparatively low amounts of an anti-TLR2 antibody completely blocked MALP-2-induced endothelial cell activation, confirming that TLR2 is on the surface, where it can be reached by the antibody.
Our observation that the anti-TLR4 antibodies did not block LPS-initiated activation could have been due to several possibilities; that the LPS was contaminated with TLR2 agonists, that the anti-TLR4 antibodies were inactive, or that HCAEC use some receptor other than TLR4 to respond to LPS. The inability of a blocking anti-TLR2 antibody to block LPS-initiated activation excluded TLR2 agonist contamination of the LPS preparation being responsible for HCAEC activation (Fig. 4). In Table 1, we confirmed over a broad range of LPS and HTA405 concentrations the inactivity of this mAb to block LPS-dependent HCAEC activation. The functional activity of the anti-TLR4 antibodies was confirmed in monocytes (Fig. 4), in THP-1 cells, and in HeLa cells transiently transfected with human TLR4 (not shown). We were not able to confirm data by others that show almost complete inhibition of LPS induced (100 ng/ml) CD62E expression in HUVEC by comparatively low concentrations of HTA125 (36). In our studies using HUVEC instead of HCAEC, anti-TLR4 antibodies were also ineffective in blocking the response to 10 ng/ml LPS (E. coli O55:B5) (data not shown). Finally, endothelial cells derived from TLR4 –/– C57/BL6 mice were completely unresponsive to LPS compared with wild-type control cells. We also found that Compound 406, a synthetic tetraacylated bisphosphate precursor of E. coli lipid A that blocks the effects of LPS on human TLR4/MD-2 (37, 38), inhibited LPS-induced HCAEC activation (data not shown), as previously also shown by Zeuke et al. (39). In contrast to antibodies, Compound 406 can enter cells like LPS. This finding further supports the involvement of TLR4 and MD-2 in HCAEC activation by LPS (37, 38). In contrast to anti-TLR4, anti-LBP (mAb 2B5) and anti-CD14 (mAb 28C5) antibodies did abolish LPS activity; 2B5 inhibits binding of LBP to LPS, and 28C5 inhibits the formation of LPS·CD14 complexes (29, 40). These antibodies inhibit extracellular events, which are also essential for LPS-dependent intracellular activation under our conditions. Our observation here (Fig. 4 and Table 1) and elsewhere (41), that anti-TLR4 antibodies are not as effective as anti-CD14 even in monocytes, suggests that some activation of monocytes could be occurring intracellularly as well as at the surface. Taken all together, these data confirm the unique role of TLR4 for LPS signaling and lead us to conclude that the TLR4 antibodies are ineffective because they cannot reach their target, which is located and functions intracellularly in HCAEC.
Hornef et al. have similarly concluded that TLR4 resides and functions intracellularly in immortalized murine intestinal epithelial cells (27). How TLR4 and TLR2 localization is controlled is unclear. Some workers have suggested an involvement of gp96 in intracellular retention of TLR1, TLR2, and TLR4 (27, 42), without providing an express mechanism. We confirmed that the cells we studied express gp96, at least at the RNA level. Whatever the mechanism controlling TLR localization, it must be both cell type specific, because endothelial/epithelial cells differ from monocytes, and TLR specific, because TLR2 can differ from TLR4.
Cognizant that LPS would need to be internalized by HCAEC to activate TLR4, we investigated requirements for LPS internalization in these HCAEC. We found that there was a basal level of uptake of either LPS or preformed LPS·CD14 complexes that could be enhanced by LBP. Both the basal and LBP-stimulated uptake were sensitive to cytochalasin D and wortmannin, suggesting that receptor-mediated endocytosis was involved (data not shown) (43). The finding that LBP is also taken up into the cells with LPS or LPS·sCD14 complexes suggests the existence of intracellular LPS complexes. In fact, these complexes were seen to colocalize with intracellular TLR4 (Fig. 7). This was not observed in the absence of sCD14, where LPS or LPS·LBP complexes are internalized without stimulating the cells. Moreover, upon LPS stimulation, the TLR4 signaling adaptor molecule MyD88 is recruited to a perinuclear region, suggesting activation of intracellular TLR4 in HCAEC.
Because LBP enhanced the uptake of stimulatory LPS·CD14 complexes, it seemed likely that LBP would also enhance HCAEC activation. This was observed to be the case at low LPS concentrations and suggests again intracellular TLR4 function, because stimulation of LPS uptake resulted in cell activation. At higher LPS·sCD14 concentrations, the LBP-independent uptake is apparently sufficient to allow activation of the cells. This observation fits with the general concept that LBP is not absolutely required for LPS-dependent cellular activation (30). In the past, the supportive role of LBP for cell stimulation was thought to be limited to its role in forming LPS·sCD14 complexes. In this work, the role of LBP in enhancing activation is also dependant on its novel role in enhancing uptake. Presumably this could also be observed with the epithelial cells studied by Hornef et al. under the appropriate circumstances (27, 28). Interestingly, in HCAEC LBP-enhanced uptake of LPS·CD14, complexes appeared to reach lower maximal levels of LPS internalization than of LPS alone. Internalized LPS complexed to LBP without sCD14 does not stimulate HCAEC, thus LBP also facilitates LPS clearance from the circulation. This phenomenon is probably responsible for the observation that very high levels of LBP protect mice from endotoxic shock (44).
Preliminary experiments to determine the receptors involved in uptake included inhibition studies with polyanionic and polycationic substances. Our data demonstrate that the LBP-dependent LPS uptake, in contrast to the basal LBP-independent LPS uptake, involves a scavenger receptor pathway. It seems that LBP-bound LPS is taken up by scavenger receptors in preference to LPS itself. Scavenger receptors that bind LPS and which can be blocked by polyanions include at least scavenger receptor A (45), CLA-1 (33), and CL-P1 (46). Expression of mRNA for CLA-1 and CL-P1 could be detected in our HCAEC (data not shown); whether one of them or some other scavenger receptors play a primary role in HCAEC activation by low endotoxin is as yet unclear. Unfortunately, both dextran and PIA stimulated HCAEC
independently of LPS and thus their potential as inhibitors of LPS-dependent activation could not be assessed.
Atherosclerosis is a chronic inflammatory disease of the arteries, including the endothelial cells, and low LPS has been suggested to be an etiologic agent (4). Low levels of circulating LPS are found even in healthy human subjects. One is tempted to speculate that TLR4 is not surface expressed in HCAEC to protect the cells from continuous activation by removing TLR4 from exposure to every circulating LPS molecule. Endothelial cells are generally less sensitive to LPS than monocytes (47), and the intracellular location of TLR4 may be responsible for this. Current literature has focused on the correlation between CRP as an inflammatory marker of risk for cardiovascular disease. It is our prejudice that CRP is unlikely to play an etiologic role in atherosclerosis, although its clinical utility as a risk indicator seems clear (48). On the other hand, LBP is also an acute phase reactant (12) and, according to these results, is likely to have an important role in initiating inflammatory responses in the vasculature in response to low, chronic LPS. Perhaps plasma LBP levels should be monitored as well as CRP. ACKNOWLEDGMENTS
This work was supported in part by grants from the Max Kade Foundation, New York, and the Austrian Science Fund (FWF project number J2310-B05) to S. Dunzendorfer. Also supported by HL-23584 and California TRDRP 11-RT-0073 to Peter S. Tobias. This is publication #16053-IMM from the Scripps Research Institute, La Jolla, CA.
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Received December 10, 2003; accepted March 31, 2004.
Effects of HTA405 on HCAEC and monocyte activation by LPS at various LPS and mAb concentrations
HCAEC
mAb HTA405 (μg/ml) MCF (units) 0 1 10 50 0 9.56 8.63 7.44 9.14 0.1 13.93 12.11 10.12 11.04 1 59.58 51.71 46.71 49.63 10 383.5 373.1 322.2 346.7
L P S
(n g /m l ) 100 768.3 804.9 790.3 767.3
Monocytes
mAb HTA405 (μg/ml) IL-8 (pg/ml) 0 1 10 50 0 43.7 57.9 58.4 53.6 0.1 235.3 216.3 112.8 109.4 1 598.4 617.6 352.6 339.0 10 628.7 441.7 420.1 392.2
L P S
(n g /m l ) 100 803.4 1028.5 732.1 680.9
Figure 1.FACS analysis of HCAEC and monocyte TLR2, TLR4, and CD14 surface expression. HCAEC were stimulated with LPS (10 ng/ml) and IFNγ (250 U/ml) or TNF (10 ng/ml) and IFNγ (250 U/ml) for 12 h. Thereafter, cells were stained with anti-TLR4 HTA125 or anti-TLR2 2392 monoclonal antibodies or isotype-matched control mouse IgG (each 10 μg/ml). The same antibodies were used in unstimulated monocytes and transfected HCAEC. Surface CD14 was visualized using an anti-CD14 mAb (M5E2). Shown is a representative result out of many experiments using a variety of anti-TLR4 antibodies. The number of positive stained cells is given as percent of all recorded cells. mRNA levels in stimulated and unstimulated HCAEC and in monocytes were determined by RT-PCR. GAPDH served as control for both TLR2 and TLR4 mRNA. To exclude genomic DNA contamination in the PCR templates from HCAEC and monocytes, the
reverse transcription reaction was performed also in the absence of reverse transcriptase (RT–).
Figure 2.RT-PCR analysis of unstimulated and stimulated HCAEC. Cells were stimulated with Escherichia coli LPS (O55:B5; 10 ng/ml) for 12h. cDNA was reverse-transcribed from 1μg total RNA and amplified by 30 cycles under standard PCR conditions. CD14 and LBP cDNA was used for control. mRNA levels of gp96 were investigated in three different HCAEC preparations and in monocytes. Products were separated in 1.2% agarose gels and visualized with
ethidium bromide.
Figure 3.3D confocal microscopy of HCAEC (A–D) and monocytes (E). Magnification 600×. A) TLR4, red; VE-cadherin (CD144) membrane staining, green; nucleus, blue. Note that CD144 as an exported protein can be found also intracellularly.B) TLR4, red; CD144, green membrane staining; Golgi, green nucleus, blue. C) 3D volume view of 20 optical sections. Cells were stimulated with LPS (10 ng/ml) for 4 h. TLR4, red; CD106, green; nucleus, blue. The “cap” of the cell has been cut off optically and enables a view inside the cell. CD106 is found on the plasma membrane and inside in the cytoplasm. TLR4 is seen only intracellularly. The yellow is not colocalization but an optical bias due to the volume view shown (depth of 20 optical sections). D) TLR4, red; F-actin staining, green; nucleus, blue.E) TLR4, red; mCD14,
green; merge, yellow (colocalization of TLR4 and mCD14).
Figure 4.Anti-TLR4 antibodies do not block LPS-induced HCAEC activation. TLR4 antibodies (20 μg/ml), anti-LBP (2B5; 20 μg/ml), anti-CD14 (28C5; 20 μg/ml), and anti-TLR2 (mAb 2392; 5 μg/ml) were added 30 min before cell stimulation with 10 ng/ml LPS (Escherichia coli O55:B5) and 100 ng/ml MALP-2 in HCAEC and 1 ng/ml LPS (E. coli
O55:B5) and 1 ng/ml MALP-2 in monocytes for a further 4 h. Activation status of HCAEC was quantified by measuring CD62E surface expression (given as mean channel fluorescence; MCF) and in monocytes by measuring IL-8 levels (pg/ml) in the medium. Data are given as means ±SE from at least five independent experiments. **P < 0.01 (Mann Whitney U
test).
【权威疗法】--PLA生物细胞免疫激活疗法 “PLA生物细胞免疫激活疗法”运用现代纳米技术，萃取出纳米小分子“PLA生物细胞”，发现该分子细胞可将发炎增厚的组织溶解代谢掉，运用到治疗风湿方面，将大量PLA生物细胞通过特异通道到达病灶部位，通过预处理方案中的超大剂量的生物细胞破坏和阻断患者体内所有自身免疫T淋巴细胞克隆和记忆细胞营养供给，清除T淋巴细胞使其失去活性并排出体外。 并能有效阻滞相应神经，使椎间隙拉宽，迅速减轻疼痛，同时在修复免疫功能分子的作用下恢复弹性，承受受压能力。由于输入的PLA生物细胞含有大量营养神经细胞及清除无菌性炎症的药物，可使受压损伤的神经细胞在3-5天内重新再生，使局部创面得到愈合，使由于压迫刺激导致的神经根及椎管内水肿得到消退，疼痛症状消失。 由于PLA生物细胞免疫激活疗法治疗风湿病，不经过血管，而是运用超导向活检实施治疗，比其他方式更容易到达病变位置，药物在病变局部高度集中，随着药物浓度升高，疗效越显着。由于在病变部位进行治疗，所以药物对病变位以外的周围组织影响甚小，避免了全身用药的负面危害，大大减少了药物本身的毒副作用。 【技术优势】--PLA生物细胞免疫激活疗法 (1)、不开刀、不手术、不住院。避免传统开刀手术带来的大创伤、易感染、难除根的弊端。 (2)、避免吃药带来的副作用。避免了患者长期用药所造成的心脏、肾脏、肠胃等损伤出现。 (3)、直达病灶除病根，预防复发。直接清除炎性病原体，修复受损骨组织，提升免疫机能。 (4)、治疗更快捷、效果稳定持久。30分钟就可实现一次性治疗，防止了骨组织再次受破坏。 (5)、真正的“绿色”治疗方式。对关节周围正常组织无损伤，保持整个关节功能的完整性。 【治疗步骤】--PLA生物细胞免疫激活疗法 1、精准定位：通过影像学仪器，针对患者病情的个体差异，进行病灶的准确定位。 2、靶向介入病灶点：通过纳米技术，提取PLA生物细胞迅速分布于病灶部位，直达病灶除病根。 3、清理T淋巴细胞：PLA生物细胞到达病灶，分解大量免疫因子和修复因子，能够迅速杀灭病变的T 淋巴细胞并清理出体外。解除受到炎性病原体浸泡的关节滑膜及软骨组织，使疼痛僵硬状态随着炎性物质的消除而消失。 4、修复软骨恢复免疫平衡：快速修复受损组织，防止骨质遭到破坏，同时促进健康滑液再生，使坏死、病变组织得以修复并及时的生出新的骨组织，从而恢复正常生理功能、提升免疫力、调节抗体平衡、预防病原体再次入侵的目的。 【显著疗效】--PLA生物细胞免疫激活疗法 1.30分钟止疼痛。
小鼠冠状动脉内皮细胞 小鼠冠状动脉内皮细胞产品说明： 为使客户能尽快开展实验，派瑞金发货的原代细胞均处于对数生长期，且每次发货为汇合率达到70%的细胞，收到细胞后即可开展实验。 派瑞金提供的小鼠冠状动脉内皮细胞取自新鲜的组织，按照标准操作流程分离培养。研发的小鼠冠状动脉内皮细胞完全培养基能提供细胞最佳的生长条件，降低杂细胞污染，保证不同批次间细胞质量的稳定。 同时，派瑞金还建立了严格的细胞鉴定流程，所提供的原代细胞均需经过细胞类型特异性标记物、细胞形态学等检测，保证细胞纯度在90%以上；同时也需经过微生物检测，保证不含有HIV、HBV、HCV、支原体、真菌及其他类型的细菌。 注意事项： 1. 收到细胞后首先观察细胞瓶是否完好，培养液是否有漏液、浑浊等现象，若有上述现象发生请及时和我们联系。 2. 仔细阅读细胞说明书，了解细胞相关信息，如细胞形态、所用培养基、血清比例、所需细胞因子等。 3. 请客户用相同条件的培养基用于细胞培养。培养瓶内多余的培养基可收集备用，细胞传代时可以一定比例和客户自备的培养基混合，使细胞逐渐适应培养条件;建议使用派瑞金的完全培养基。 4. 建议客户收到细胞后前3天各拍几张细胞照片，记录细胞状态。 5. 该细胞只能用于科研，不得用于临床应用。 小鼠冠状动脉内皮细胞产品简介： 产品名称：小鼠冠状动脉内皮细胞（Mouse Coronary Artery Endothelial Cells）组织来源：小鼠冠状动脉 产品规格：5×105cells/25cm2培养瓶 小鼠冠状动脉内皮细胞简介： 冠状动脉是供给心脏血液的动脉，起于主动脉根部，分左右两支，行于心脏表面。冠状动脉内皮细胞分离自正常小鼠冠状动脉组织，呈单层扁平分布。内皮细胞或血管内皮是一薄层的专门上皮细胞，由一层扁平细胞所组成。它形成血管的内壁，是血管管腔内血液及其他血管壁（单层鳞状上皮）的接口。内皮细胞是沿着整个循环系统，由心脏直至最少的微血管。 本公司生产的小鼠冠状动脉内皮细胞采用混合酶消化获得，细胞总量约为 5
生物免疫疗法 1、CIK细胞简介 细胞因子诱导的杀伤细胞(cytokine induced killer cell，CIK)是一种新型的免疫活性细胞，它是将人外周血单个核细胞在体外用多种细胞因子共同培养一段时间后获得的一群异质细胞，由于该细胞群中多数细胞同时表达CD3和CD56两种膜蛋白分子，故又被称为NK细胞样T淋巴细胞。CIK细胞具有增殖速度快、杀瘤活性高、杀瘤谱广及非MHC限制性杀瘤的优点。目前，CIK疗法被认为是新一代抗肿瘤过继免疫治疗的首选方案。 2、CIK细胞的抗肿瘤机制 （1）CIK细胞对肿瘤细胞的直接杀伤 CIK细胞能分泌穿孔素(proforin，PFP)和颗粒酶（granzyme），通过穿孔素/颗粒酶途径介导杀伤靶细胞。 （2）CIK细胞活化后产生大量细胞因子，发挥抑瘤杀瘤作用 CIK细胞活化后产生大量的炎性细胞因子，如IL-2、IL-4、IL-10、IFN-γ、TNF-α等，不仅对肿瘤细胞有直接抑制作用，还可通过调节机体免疫系统反应性间接杀伤肿瘤细胞。 （3）诱导肿瘤细胞凋亡 CIK细胞能活化肿瘤细胞凋亡基因，并且CIK细胞内FLIP、Bcl-2、Bcl-xl、DAD1和survivin 等抗凋亡基因表达上调。这些原因共同导致了CIK细胞可以通过Fas/FasL信号通路诱导肿瘤细胞的凋亡，从而对肿瘤进行有效的杀伤。 图1 CIK细胞杀伤肿瘤细胞途径与机制
图2 CIK细胞的抗肿瘤机制 3、CIK细胞的临床应用 （1）适应征 近年的临床研究显示，CIK细胞在临床治疗的早、中、晚期恶性实体瘤病例和白血病中均有较好的疗效。CIK细胞生物免疫治疗适用于： 病人在手术切除原发肿瘤后+CIK 化疗、放疗间期结合CIK细胞免疫支持治疗不仅能有效杀灭残留癌细胞，而且可以提高放化疗病人的自体免疫力，起到支持治疗的目的； 微创介入、氩氦刀、Cyber刀等治疗的同时或治疗后辅以CIK细胞治疗疗效更佳； 部分晚期癌症患者，自身体力条件不允许行放化疗的患者； 暂时不宜做手术、介入或其他治疗的肿瘤患者也可先进行CIK细胞治疗，提高身体机能状况，改善生活质量，争取其他治疗机会。 对部分化疗多药耐药瘤株同样具有强大杀伤作用（如p-gp阳性乳腺癌） （2）治疗策略
——干细胞免疫疗法是世界上的终端疗法 当年一个全民补钙的理念，今天“双青胶囊”使补骨髓成为现实，全民补骨髓的趋势刚刚兴起，将给人类带来健康活力。 树老干枯，人老髓衰，骨髓饱满疾病全无，骨髓流失器官衰老，骨髓免疫寿命延长，年轻的骨髓让生命返老还童！骨髓是干细胞的种子，免疫细胞的源泉，“双青胶囊”能透过骨髓滋养孔对骨髓进行全方位的滋养调理，三个月等同全新的骨髓再造，全新的骨髓能激活人体静止的干细胞，提高造血干细胞的数量，修复免疫系统，主动发挥免疫调节作用，根本改变人体内部环境，从而起到对疾病的治疗作用，这是医疗史上的一场革命，也是世界上的终端疗法——干细胞免疫疗法。 一、干细胞疗法——现今世界上的终端疗法 干细胞疗法也叫免疫疗法。它的基础概念是——骨髓干细胞。这种细胞通过分化再生各种不同的细胞。简单的说，人体内骨髓的增多，能使造血干细胞数量提高，因此骨髓的再生和增强对于维持人体生命和免疫力都十分重要。骨髓再生能整体调节神经系统、免疫系统、内分泌系统。使这三大系统有效的增强调控能力。促使消化系统、循环系统、骨骼系统、呼吸系统、皮肤系统、生殖系统等六大系统自然恢复功能。均可使许多疾病不治自愈和内环境的稳定。使肌体健康生长和生存。干细胞作为一类未分化细胞和原始细胞具有自我复制能力，在一定条件下，干细胞可以定向分化肌体内的功能细胞，形成任何类型的组织和器官具有可朔性。因此被称为源泉细胞、万能细胞，它能抑制细胞变异，并能修复、排除变异细胞、再生、分化新的细胞。 当骨髓生成神经干细胞时，它可以治疗帕金森氏病症、老年性痴呆症、脊柱侧弯硬化及外伤所致的脊柱损伤、中枢神经肿瘤等；生成胰腺干细胞可治疗胰岛素依赖型糖尿病及其它型糖尿病；生成肝脏造血干细胞时可治疗慢性肝炎肝腹水肝硬化；生成角膜干细胞时可以治疗各种眼病，能使眼角膜再生。干细胞疗法应用于各种疾病的预防和治疗必将成为人类健康长寿的福音。 二、骨髓——免疫细胞的孵化器
iNOS在不同年龄正常和冠状动脉粥样硬化血管内皮细胞中的表达 发表时间：2018-11-22T15:10:00.447Z 来源：《中国医学人文》(学术版)2018年5月上第9期作者：苏琳 [导读] 如何通过iNOS抑制剂的使用，抑制iNOS 的表达，减少NO的生成，减少急性心肌坏死和凋亡，仍需要循证医学的不断探索。 浙江大学医学院附属邵逸夫医院全科医学科浙江杭州 310000 【摘要】目的：研究不同年龄段正常人群和冠状动脉粥样硬化患者血管内皮细胞中诱导型一氧化氮合酶（iNOS）的表达，验证iNOS 对冠状动脉粥样硬化疾病的作用。方法：每个年龄组（20-40岁、41-50岁、51-60岁、61-70岁、70岁以上）取心脏左前降支中的一段，每组正常人群及患者各2个。制成石蜡切片，通过免疫组化染色，观察结果。结果：iNOS在冠状动脉粥样硬化患者血管内皮细胞中的表达明显高于正常人群血管内皮细胞中的表达。结论：iNOS在冠状动脉粥样硬化患者血管内皮中的表达高，说明iNOS在冠状动脉粥样硬化的发生发展中起重要作用。 【关键词】iNOS；冠状动脉粥样硬化；内皮细胞 冠心病是动脉粥样硬化导致器官病变的最常见类型，也是严重危害人类健康的常见病。动脉粥样硬化的病理改变是内皮舒张因子产生减少及内皮细胞受损，平滑肌细胞增殖、脂质沉积，从而引起血管壁增厚乃至管腔狭窄，血管张力增加。一氧化氮（nitric oxide，NO）是体内的主要舒血管活性物质，具有维持血管张力，调节血压，扩张冠状动脉，调节心肌收缩与舒张[1]，增加心肌供血等作用。在体内，NO 主要由一氧化氮合酶（nitric oxide synthasen，NOS）催化L-精氨酸产生。NOS在体内广泛分布[2]，其中血管内皮细胞是最为集中的部位[3]。NOS 主要有三种不同的亚型[4]：神经型NOS（nNOS）、诱导型NOS（iNOS）及内皮细胞型（eNOS）。当内皮细胞受到炎症等细胞因子刺激时，iNOS 会受刺激诱导而开始表达[5]。iNOS一经表达，即具有高度的活性，且持续时间长，催化生成较高浓度的NO，参与心血管的许多病理过程[6、7]。本次实验用免疫组化Envision二步法检测iNOS在冠状动脉血管内皮中的表达，验证iNOS对冠状动脉粥样硬化疾病的作用。 1 材料 1.1 冠状动脉样本取自浙江大学司法鉴定中心。 1.2 免疫组化试剂一氧化氮合成酶1型，编号BA0360购于武汉博士德生物工程有限公司；羊抗免IgG抗体-HRP多聚体，编号PV-6001，购于北京中杉金桥生物技术有限公司；浓缩型DAB试剂盒，编号ZLI-9031/9032/9033，购于北京中杉金桥生物技术有限公司。 1.3 其它试剂和材料不同浓度二甲苯溶液、不同浓度酒精溶液、PBS缓冲液、苏木精染液、0.01mol/L枸橼酸钠缓冲液、3%H2O2溶液、多聚左旋赖氨酸载玻片、树胶、微量加样器、冰箱、湿盒、切片架、显微镜。 2 方法 2.1 冠状动脉材料准备挑选出符合要求的非冠状动脉粥样硬化和冠状动脉粥样硬化的尸体标本各10例，然后找到心脏冠状动脉左前降支，切取约2-4mm的一段血管，制成切片。 2.2 切片制成后，于60℃烘箱烘烤切片4-6小时。 2.3 HE染色 切片脱蜡将切片依次浸入：二甲苯I 10分钟，二甲苯II 5分钟，无水乙醇I 5分钟，无水乙醇II 5分钟，95%乙醇5分钟，90%乙醇5分钟，80%乙醇5分钟，水洗2分钟。 染色苏木精染色10分钟，水洗1分钟，1%盐酸乙醇分化数秒，再用水洗1分钟。用0.2%氨水返蓝约30秒，水洗1分钟。伊红染色5分钟，快速用水洗去染料。 脱水、透明、封固将切片依次浸入：80%乙醇20秒，90%乙醇20秒，95%乙醇I 3分钟，95%乙醇II 3分钟，无水乙醇I 5分钟，无水乙醇II 5分钟，二甲苯I 3分钟，二甲苯II 3分钟，二甲苯III 3分钟。 最后用中性树胶封片。 2.4 免疫组化法染色采用免疫组化envision二步法： 切片脱蜡和水化将切片放入60℃恒温箱中烘烤20分钟，取出后将组织切片依次置于下列溶液中浸泡：二甲苯I 10分钟，二甲苯II 10分钟，无水乙醇5分钟，95%乙醇5分钟，80%乙醇5分钟，70%乙醇5分钟。取出后用PBS洗三次，每次3分钟； 封闭内源性过氧化物酶活性将切片在3% H2O2溶液中浸泡15分钟。取出后再在PBS中洗一次，3分钟； 抗原修复用电磁炉加热水浴锅，水浴加热0.01ml/L枸橼酸钠缓冲液（pH 6.0）至95±3℃，将切片放入缓冲液中，稳定温度保持30分钟。结束后连枸橼酸钠缓冲液一起从水浴锅中取出，放在室温下自然冷却； 滴加一抗将自然冷却的切片用PBS洗两次，每次3分钟。然后滴加1：100稀释的一抗，稀释后抗体浓度为2μg/ml，液体完全盖过组织块，整个操作不能干片。滴加完一抗以后，抗ETA和抗ETB两套抗体的切片必须分开操作，不能相互接触。将滴加完一抗的切片放入湿盒切片架，放入4℃冰箱中过夜（约20小时）； 滴加二抗取出过夜后的切片，待其恢复至室温后，用PBS溶液洗三次，每次3分钟。滴加二抗，即山羊抗免IgG抗体-HRP多聚体，湿盒孵育30分钟，取出，PBS洗三次，每次3分钟； DAB显色按DAB试剂说明书，在进行DAB染色前数分钟配好DAB溶液。在水槽边向切片滴加DAB溶液，待颜色微黄后立即用水冲去多余染液； 苏木精复染将切片浸入苏木精染液中5分钟，取出用水洗净； 封片中性树脂封片。 3 结果 判定标准细胞浆被染成棕黄色作为阳性细胞判定标准。阳性细胞< 5 %为浅黄色（±）；5 %～25 %之间为棕黄色（+）；26 %～50 %之间为中度棕色（++）；> 50 %为深棕色（+++）。 从表1可看出：正常血管内皮和冠状动脉粥样硬化血管内皮中iNOS的表达差异明显。冠心病患者冠状动脉血管内皮细胞中iNOS阳性表
生物免疫治疗的优缺点 癌症是一种严重威胁着我们身体健康的疾病，也是一种致命率较大的疾病。当我们患上这种疾病的时候，就代表着我们的生命即将走向尽头，开始了倒计时的日子。癌症不仅仅是一种致命性较大的疾病，它还会让很多的症状出现在我们的身上，让我们的身心健康都承受着巨大的压力。 在医疗界中，因为癌症死亡的人不在少数。即使看惯了死亡的医护人员，看到癌症对患者的折磨，还是会产生恐慌感。癌症会在患者的身体中出现很多的症状，尤其是疼痛这种症状。疼痛会贯穿癌症患者的整个治疗过程中，有些患者承受不住疼痛的折磨，甚至会产生轻生的念头，选择放弃自己的生命。为此，癌症治疗成为了一件至关重要的事情。 癌症一般都会使用手术治疗、放射治疗、化学治疗这三种治疗方式。但是这三种治疗方式却存在着一定的弊端。他们都会在患者的身体中出现很多的副作用，让患者苦不堪言。 为了弥补这三种癌症的治疗方式，在医疗人员的研究下，生物免疫治疗技术出现了。它是一种新兴的癌症治疗技术，可在一定的程度上缓解癌症患者的痛苦。 生物免疫治疗技术，是一种应用最广、最成熟的肿瘤生物治疗技术，目前临床上常用的是多细胞免疫治疗即自体免疫细胞治疗技术(用自己的细胞治自己的病)，它是提取患者自身的免疫细胞进行体外培养，然后再回输到患者体内，用自己的细胞治疗自己的病，不仅无明显毒副作用，还能改善免疫功能，故不会发生排异,治疗更简便、安全。 多细胞免疫治疗是采取患者外周血，经过培养增殖，培养出具有识别和杀伤肿瘤的DC细胞和直接杀伤肿瘤的CIK细胞，还有识别杀伤癌细胞能力最强的高纯度的NK细胞，以及NKT细胞和CD3AK细胞，再以打点滴的方式回输患者体内，五种细胞强强联合，能清除体内不同部位的微小残留病灶，有效防止肿瘤复发与转移。多细胞免疫治疗技术联合手术、化疗和放疗综合治疗，能起到良好的临床疗效，对不同肿瘤实施“个性化”免疫细胞治疗方案，进一步提高了治疗效果。这种联合应用多种免疫细胞实施个性化治疗的技术与方案将成为未来肿瘤治疗的发展趋势。 生物免疫治疗技术的优点： 效果确切，有效率高:对有些癌症，有效率高达90%。 无放、化疗毒副作用，病人不痛苦，耐受性好，杀瘤特异性强。 能够激发全身性的抗癌效应，对多发病灶或转移的恶性肿瘤同样有效。 可以帮助机体快速恢复被放、化疗破坏的抗癌免疫系统，提高远期抗癌能力。
1例成人朗格汉斯细胞组织细胞增生症患者的护理 朗格汉斯细胞组织细胞增生症(langerhanscellhistiocyto sis,LCH)既往被命名为组织细胞增生症,是一种病因不明的免疫失控性疾病。目前多认为是一种非肿瘤性的朗格汉斯细胞异常增生,此种增生可能是由于内源性或外源性刺激导致免疫调节功能紊乱所致,临床较罕见,常采用化疗、激素、放疗方法进行单独或联合治疗[1]。我科于2009年9月收治了1例肺、肝、脾、甲状腺及淋巴结等多器官受累的成人朗格汉斯细胞组织细胞增生症患者,经抗炎、化痰、保肝、利胆及(10%葡萄糖250ml+长春新碱4mg静滴,第1d;生理盐水250ml+环磷酰胺1g静滴,第1d;强的松40mg口服,第1~7d,简称VCP方案)化疗后,患者情况明显好转,予以出院。现将护理体会报道如下。 1病例介绍 患者,男,31岁,因确诊朗格汉斯细胞组织细胞增生症3年余,咳嗽、咳痰20余天,自服沐舒坦无效!,于2009年9月15日由门诊收住入院。患者于3年前胸腔镜下肺组织病理活检确诊为朗格汉斯细胞组织细胞增生症,给予强的松口服治疗1年,后因发现颈部淋巴结肿大,行淋巴结活检及肝脏彩超及肝功能等检查后诊断为肝脏、淋巴结朗格汉斯细胞浸润及甲状腺功能减低,肝功谷丙氨酸转氨酶(ALT)、谷草氨酸转氨酶(AST)明显升高,在外院予长春新碱+环磷酰胺化疗5次。入院查体:全身皮肤及黏膜重度黄染、巩膜重度黄染。 肝功能:总蛋白57g/L,白蛋白31g/L,球蛋白26g/L,丙氨酸氨基转移酶 276U/L,门冬氨酸氨基转移酶216U/L,碱性碱酸酶2544U/L,-谷氨酰转肽酶216U/L,乳酸脱氨酶(LDH)262U/L。胸部CT示:肺部及脾脏表现符合组织细胞增生症;肝脏核磁共振(MRI):肝脾肿大,肝内多发炎性结节,肝胆管炎。入院后予特治星抗炎、吉诺通化痰、甘利欣保肝及补充白蛋白、利胆退黄疸治疗后予VCP 方案化疗,复查肝功能:总蛋白59g/L,白蛋白33g/L,球蛋白32.3g/L,丙氨酸氨基转移酶182U/L,门冬氨酸氨基转移酶118U/L,碱性碱酸酶920U/L,-各氨酰转肽酶1109U/L,乳酸脱氨酶(LDH)207U/L,全身皮肤及黏膜、巩膜黄染较前明显减退、病情平稳,于2009年10月11日出院。 2护理 2.1心理护理 患者为年轻男性,病程长,所患疾病临床少见,经反复多次对症支持治疗及化疗,效果欠佳,病情持续加重,出现多器官受累,入院时情绪抑郁。护士加强巡视,做好主动介绍,关心帮助患者,详细介绍治疗方案及药物主要作用,及时告知治疗所取得的进展,使患者能够积极主动地配合治疗。根据需要,留家属陪伴,利用亲情鼓励患者树立起战胜疾病的信心。
肿瘤生物免疫疗法要做几个疗程 生物免疫疗法一般需要做几个疗程是因人而异的，一般早期的肿瘤患者及时到院治疗，一到两个疗程就可达到预期的效果，甚至实现临床治愈，只需后期定期的复查既可；而对于一些中晚期的恶性肿瘤患者来讲，一到两个疗程可能起到的就是控制肿瘤的发展，如果想实现理想的治疗效果，就需要采取一些综合治疗方案，或是增加一到两个疗程的生物治疗，所以具体情况还要根据您目前的身体状况进行确定。不管治疗需要几个疗程，患者都不会有任何不良反应。 肿瘤生物免疫治疗，重启肿瘤患者免疫系统 肿瘤生物免疫治疗的诞生填补了手术、放化疗等常规疗法的不足，其不但具有清除体内不同部位的微小残留病灶，防止肿瘤复发与转移的作用，而且对病人受损的免疫系统又能起到恢复与重建的疗效。 生物免疫治疗应用免疫细胞群谱广，可以有针对性地联合应用多种免疫细胞，实现对不同肿瘤实施“个性化”免疫细胞治疗方案，进一步提高肿瘤治疗效果。手术后的肿瘤患者使用生物免疫治疗可以清除体内散在癌变细胞，预防多种肿瘤术后的复发和转移，如肝、肾部位肿瘤；食管、胃、肺、肠、乳腺等肿瘤患者在生物免疫治疗与放化疗结合使用的治疗中显示出很好的协同作用和疗效维持效果，大大改善了肿瘤患者的身体状况，减轻放化疗反应；处于康复期的肿瘤患者采用生物免疫治疗进行巩固治疗，可以控制肿瘤生长，维持疗效，并改善患者生活质量，提高患者生存机会和存活时间。 生物治疗肿瘤分为两大类，DC-CIK生物治疗和多细胞(高纯度NK)免疫治疗，多细胞(高纯度NK)免疫治疗是目前肿瘤(癌症)治疗最先进的技术。
葫芦岛市中心医院肿瘤生物治疗中心为了弥补单一使用“DC-CIK细胞”抗肿瘤的缺陷，在积累了丰富的生物免疫治疗(DC-CIK疗法)经验的基础上，增加(高纯度)NK、CD3AK、NKT三种免疫细胞的多细胞治疗模式，针对不同患者、不同阶段，有选择性地运用具有特异性的靶向免疫细胞，抑制肿瘤的生长、转移、复发，并同时提高机体免疫力，可独立使用，与手术、放化疗联合治疗效果更佳。以保障患者生活质量、提高远期生存率的治疗目标来指导癌症治疗，能从患者全身特点加以考虑，而不只是着眼于癌症病灶本身，是患者最好的选择。 多细胞免疫治疗具体流程 (一)与患者沟通交流 这个过程主要是为了让医生了解患者的大概病情，从而初步判断患者是否适合肿瘤多细胞免疫治疗 (二)检查并确定治疗方案 为患者做一些常规检查，客观详细分析患者病情，查看患者是否对多细胞免疫治疗存在禁忌症，如果符合多细胞免疫治疗的各项客观条件要求，专家会给患者制定具体的治疗方案。 (三)采集外周血单个核细胞 治疗方案经患者及家属同意后，便可以进行细胞采集，提取患者80ml-100ml 血液，这个过程患者不会感到任何不适。
血管内皮细胞功能与心血管疾病关系的研究进展 摘要：血管内皮细胞（VEC）功能与心血管疾病的发生和发展密切相关, 本文综述了血管内皮细胞合成与释放的一氧化氮、内皮素、血管紧张素Ⅱ等多种生物活性物质及VEC功能紊乱与心血管疾病的关系，研究VEC功能与心血管疾病形成之间的相互关系将为心血管疾病的治疗提供新思路、新策略。 关键词：血管内皮细胞；一氧化氮；内皮素；血管紧张素Ⅱ；心血管疾病 现已证明VEC除了完成血液和组织液的代谢交换以外，还是机体最大的内分泌腺【1】，可以产生和分泌十余种生物活性物质，具有维持正常的血管张力、血液的正常状态和动态平衡等作用，并通过其屏障和分泌功能，影响着炎症反应的发生、发展，参与调节机体的免疫应答。VEC功能紊乱在高血压、心力衰竭、动脉粥样硬化（AS）等心血管疾病的发病过程中具有重要意义。 1血管内皮细胞功能【2】 VEC的功能极其重要和复杂。1维持血管内膜的光滑，防止血小板及白细胞粘附，防止有害物质侵入血管壁。2具有半透膜的作用，维持血液、组织液中物质的交换。3合成并释放多种生物活性物质，如一氧化氮（NO），PGI2,，内皮素（ET）等，调节血管张力，维持正常血压。4合成致栓及抗栓物质，维持其动态平衡，如肝素，组织型纤溶酶原激活物（t-PA）及血管性假血友病因子（vWF）等。5 合成胶
原基底膜及血管平滑肌保护层。6合成血管生长因子及血管紧张素转化酶（AEC）。7影响血管壁对脂蛋白等物质的代谢。 2血管内皮细胞功能的标志物质【3~5】 2.1 一氧化氮(NO)。NO由血管EC释放，EC以L一精氨酸和分子氧作为底物，在NO合酶作用下生成NO 继之进入邻近的平滑肌细胞，激活鸟苷酸环化酶分解GTP使c—GMP增加，导致血管平滑肌舒张。NO还有抑制血管平滑肌增殖、抗血小板聚集和抗血栓形成作用【 3.5】。基础状态下，EC作为血藏感受器，转化血液切变力的机械信号为化学剌激，促使NO释放，维持血管张力和血流量相对恒定。乙酰胆碱、5_羟色胺、P物质，缓激肽、凝血酶、腺苷、TXA2、组胺{细胞因子如白介素-1、肿瘤坏死因子以及内毒素，都能促使NO释放。 2．2 PGI2。EC通过环氧化酶及前列环素合成酶途径代谢花生四烯酸产生PGI2，再通过第二信使cAMP发挥生物学效应。凝血酶、缓激肽、组胺，腺苷、高密度脂蛋白、TXA2、白三烯、血小板生长因子、组织缺氧和血流动力学应激等，促使EC释放PGI2。PGI2和NO 协同扩张血管，防止血小板聚集和抗血栓形成【5】。 2．3ET1988年Yanagisawa【6】从猪主动脉EC中分离提纯出21 个氨基酸组成的多肽,称内皮素。ET 受体中B型主要分布在EC,激活后可使EC释放NO、PGI2,但其舒血管效应常被A型受体兴奋所致平滑肌细胞强烈而持久的收缩所掩盖。有实验表明,给大鼠持续滴注ET1 后血液浓缩,微动脉和微静脉收缩,微循环血流量减少,血栓形成;
朗格汉斯细胞组织细胞增生症的原因 文章目录*一、朗格汉斯细胞组织细胞增生症的简介*二、朗格汉斯细胞组织细胞增生症的原因*三、朗格汉斯细胞组织细胞增生症的危害*四、朗格汉斯细胞组织细胞增生症的高发人群*五、朗格汉斯细胞组织细胞增生症的预防方法 朗格汉斯细胞组织细胞增生症的简介肺朗格汉斯细胞组织细胞增多症(pulmonary Langerhanscell histiocytosis, PLCH)又名朗格汉斯细胞肉芽肿,是一类相对罕见的肺脏疾病,为一种朗格汉斯细胞增生性疾病,侵犯皮肤和皮肤外的器官。通常在青年人发病,与吸烟密切相关,大多表现为良性和迁延的病程。肺组织病理以以朗格汉斯细胞增生和浸润为特征,形成双肺多发的细支气管旁间质结节和囊腔。 朗格汉斯细胞组织细胞增生症的原因PLCH的发病与烟草暴露密切相关,绝大多数(90%-100%)的PLCH患者都有吸烟史,特别是吸烟大于20年者。过去认为男性发病率高于女性,近年的研究发现,PLCH在男女之间的发病率并没有明显的差异,这种差异可能与女性吸烟者人数上升有关。多数PLCH患者是重度吸烟者,但吸烟史较短的患者也可以发生PLCH。戒烟后PLCH患者的胸部影像可以好转,终末期PLCH接受肺移植的患者,如果继续吸 烟,PLCH可能再发。尽管PLCH发病与烟草暴露有关,吸烟量与
PLCH疾病的严重程度没有相关性。个案报道,PLCH在淋巴瘤放疗和(或)化疗后也可以发病。绝大多数PLCH是散发病例,也有个别LCH家族病例的报道,其发病与基因的相关性,尚不清楚。目前没有证据表明职业或地理因素与PLCH发病有关。除烟草外,病毒等外源性物质在PLCH的发病中有无作用,目前尚不明确。 朗格汉斯细胞组织细胞增生症的危害可并发： 1、慢性中耳炎和外耳炎:颞骨乳突和岩状部位受累所引起。 2、眼眶部位肿块可引起突眼,视神经或眼球肌受侵犯导致视力减退或斜视。 3、骨质侵犯最常见的部位是扁骨(如颅,肋,骨盆和肩胛骨)。长骨和腰椎,骶骨较少受累。长骨上的病损酷似Ew-ing肉瘤,成骨肉瘤和骨髓炎。腕,头,膝,足或颈椎骨侵犯属罕见。常有患儿家长陈述患儿早熟性出牙,实际上这是由于牙龈退缩,未成熟牙 质暴露的缘故。 4、胃肠道症状,尿崩症及甲状腺增大。 朗格汉斯细胞组织细胞增生症的高发人群青年人。 朗格汉斯细胞组织细胞增生症的预防方法PLCH是一种吸烟
免疫细胞治疗现状 一、免疫细胞治疗概念和分类 细胞治疗分为干细胞治疗和体细胞治疗（免疫细胞治疗）。前者包括ES、神经干细胞、骨髓干细胞、外周造血干细胞、间充质干细胞、脐带血干细胞、脂肪干细胞治疗等。后者体细胞治疗一般是指免疫细胞治疗。干细胞治疗是通过干细胞移植来替代、修复患者损失的细胞,恢复细胞组织功能,从而治疗疾病。 免疫细胞治疗技术是通过采集人体自身免疫细胞，经过体外培养，使其数量扩增成千倍增多，靶向性杀伤功能增强，然后再回输到患者体内，从而来杀灭血液及组织中的病原体、癌细胞、突变的细胞。免疫细胞治疗技术具有疗效好、毒副作用低或无、无耐药性的显着优势，成为继传统的手术疗法、化疗和放疗后最具有前景的研究方向之一。 免疫细胞治疗主要包括非特异性疗法LAK、CIK、DC、NK 和特异性TCR、CAR。（非特异性没有明确的免疫细胞靶点，是从整体上提高人体免疫力而达到缓解肿瘤症状，特异性治疗具有明确的靶点和机制，能通过激活或者抑制明确靶点来实现免疫系统对肿瘤的免疫激活）。 目前，国际最领先的是CAR-T细胞治疗，辉瑞、诺华等巨头与生物技术公司合作从事CAR或TCR开发，国内这块还没出成果，还处在临床试验阶段。而我国广泛使用的是非特异性细胞疗法，主要涉及CIK，DC-CIK治疗，比如双鹭药业，北陆药业，中源协和等公司已经运用于临床治疗中，还有些处在临床试验阶段。 二、传统非特异性细胞治疗 CIK细胞（细胞因子诱导的杀伤细胞），是将人体外周静脉血单个核细胞在体外用多种细胞因子共同培养而获得的一群异质性细胞，其中CD3+和CD56+双阳性细胞为其主要效应细胞，兼具T细胞特异性杀肿瘤和NK细胞非MHC限制性杀瘤的特点。目前CIK细胞主要用于手术后、放化疗后微小残留病灶的清除及调节患者免疫能力。 CIK 细胞具有提高机体免疫功能，清除肿瘤微小残余病灶，防止复发的作用，主要通过以下3 种途径发挥抗肿瘤作用。 （1）直接杀伤肿瘤细胞：CIK 细胞表面含有与靶细胞（肿瘤细胞）表面分子结合的受体，启动细胞溶解反应释放一些细胞毒颗粒或因子，从而溶解肿瘤细胞。 （2）释放细胞因子抑制或杀伤肿瘤细胞：CIK 细胞可分泌IL-2、IFN-γ、TNF-
Human Coronary Artery Endothelial Cells (HCAEC) Catalog Number: 6020 Cell Specification Endothelial cells constitute the natural interface between the blood and the underlying tissue. Changes in endothelial cell function appear to play a key role in the pathogenesis of atherosclerosis. Endothelial cells synthesize and secrete activators as well as inhibitors of both the coagulation system and the fibrinolysis system in addition to mediators that influence the adhesion and aggregation of blood platelets. Endothelial cells also release molecules that control cell proliferation and modulate vessel wall tone [1]. Human endothelial cells produce antithrombotic and thrombotic factors such as t-PA and PAI-1 and respond to TNF-alpha by modifying growth characteristics, producing cytokines such as GM-CSF, expressing ICAM-1 on the surface and producing large amounts of nitric oxide and endothelin [2]. Endothelial injury, with consequent endothelial dysfunction, is caused by percutaneous transluminal coronary angioplasty [3] and may play an important role in subsequent restenosis [4]. Many of the endothelial processes can be studied in vitro using cultured cells, and HCAEC will surely be very useful to studying and understanding the molecular mechanisms involved in coronary artery diseases. HCAEC from ScienCell Research Laboratories are isolated from human coronary artery. HCAEC are cryopreserved at passage one and delivered frozen. Each vial contains >5 x 105 cells in 1 ml volume. HCAEC are characterized by immunofluorescent method with antibodies to vWF/Factor VIII and CD31 (P-CAM) and by uptake of DiI-Ac-LDL. HCAEC are negative for HIV-1, HBV, HCV, mycoplasma, bacteria, yeast and fungi. HCAEC are guaranteed to further expand for 15 population doublings at the conditions provided by ScienCell Research Laboratories. Recommended Medium It is recommended to use Endothelial Cell Medium (ECM, Cat. No. 1001) for the culturing of HCAEC in vitro. Product Use HCAEC are for research use only. It is not approved for human or animal use, or for application in in vitro diagnostic procedures. Storage Directly and immediately transfer cells from dry ice to liquid nitrogen upon receiving and keep the cells in liquid nitrogen until cell culture needed for experiments. Shipping Dry ice. Reference [1] Elhadj, S. and Forsten, K. E. (2001) Chronic shear stress affects bovine aortic endothelial cell secreted proteoglycan production in vitro. Bioengineering Conference, Vol. 50:767-768. [2] Donnini, D., Perrella, G., Stel, G., Ambesi-Impiombato, F. S., Curcio, F. (2000) A new model of human aortic endothelial cells in vitro. Biochimie 82:1107-14. [3] Vassanelli C, Menegatti G, Zanolla L, Molinari J, Zanotto G, Zardini P. (1994) Coronary vasoconstriction in response to acetylcholine after balloon angioplasty: possible role of endothelial dysfunction. Coron Artery Dis. 5:979-986. [4] Meurice T, Vallet B, Bauters C, Dupuis B, Lablanche JM, Bertrand ME. (1996) Role of endothelial cells in restenosis after coronary angioplasty. Fundam Clin Pharmacol. 10:234-242.
关键词组织细胞增多症；朗格汉斯细胞 中图分类号R551.1 文献标识码 A 文章编号1001-7399(2000)02-0149-05 朗格汉斯细胞组织细胞增生症(Langerhans cell histiocytosis，LCH)是外检中比较少见的疾病。由于对它的认识不足，易致漏诊和误诊；另一方面由于对它的认识还在不断深化，以致无论在诊断名称的选用和对其本质及预后的判断上均存在混乱。现对其作简介如下。 1 “组织细胞”的起源与分类 从免疫学的角度看，“组织细胞”是能捕捉、加工、处理抗原，并将抗原提呈给抗原特异性淋巴细胞的一类免疫辅佐细胞(accessory cell, A细胞)，称抗原提呈细胞(antigen-presenting cell, APC)。它包括单核巨噬细胞系统(mononuclear phagocyte system, MPS)和树突状细胞系统(dendritic cell system, DCS)。 目前认为，抗原提呈细胞的两大系统均起源自骨髓CD34阳性干细胞，它先发育为粒细胞、红细胞、单核/巨噬细胞及血小板克隆形成单位(colony-forming unit granulocyte-erythroid-monocyte/macrophage-megakaryocyte, CFU-GEM-M)，进而发育为粒细胞-巨噬细胞克隆形成单位(CFU-GM)。后者可分化为粒细胞和单核巨噬细胞，或发育为树突状-朗格汉斯细胞克隆形成单位(CFU-dendritic-Langerhans cell, CFU-DL)。CFU-DL再分化为树突状细胞、朗格汉斯细胞(CD1a阳性)及单核/巨噬细胞(CD1a阴性)〔1〕。 1.1 单核巨噬细胞系统单核巨噬细胞体积大，表面有较多的嵴状突起，并有多种受体，如FcγR、C3bR、吸附淋巴细胞受体、细胞因子受体、清道夫受体等。胞质丰富，伊红染，内有较完善的细胞器，尤其是溶酶体与线粒体。核圆或卵圆或肾形，中位或偏位。单核细胞具有强大的吞噬功能；并能产生大量细胞因子；参予摄取、加工、处理、提呈抗原并激发免疫反应和参予免疫调节。免疫组化显示CD68(Kp-1)(+)、Mac387(+)、溶菌酶(+)、CD15(+)、LCA(+/-)。 单核巨噬细胞在体内分布：结缔组织(组织细胞)、肝(Kupffer细
血管内皮细胞与心血管疾病的关系及其研究进展 刘群峰,马 虹综述 (中山医科大学附属第一医院心内科,广东广州510089) 关键词:血管;内皮;综述文献 中图分类号:R543 文献标识码:A 文章编号:100523271(2000)022******* 血管内皮细胞(V EC)为衬贴于血管内腔面的单层扁平细胞,其表面积可达1000m2以上。V EC 分泌多种调节血管功能的活性物质,与心血管疾病有密切关系。最近已有研究试图通过干预治疗以恢复某些心血管疾病患者的内皮功能,改善预后,这是心血管疾病治疗方法中新的探索。现就V EC与心血管疾病的关系及其研究进展作一简要综述。 1 血管内皮细胞合成与释放的血管活性物质 1.1 V EC合成与释放的缩血管活性物质 V EC 合成与释放的缩血管活性物质包括内皮素(ET)、内皮依赖性收缩因子(EDCF)、前列腺素H2(PGH2)、血栓素A2(TXA2)、血管紧张素 (A g )等。其中ET可分为ET21,ET22,ET233种。ET21是目前发现的最强烈的缩血管活性多肽,它通过激活磷脂酶C起到有丝分裂原的作用,刺激血管平滑肌细胞(V S M C)内c2fo s,c2m yc原癌基因的表达,增加V S M C的DNA合成,促进V S M C的增殖,因此在动脉粥样硬化形成的过程中起重要作用[1]。 1.2 V EC合成与释放的舒血管活性物质 V EC 合成与释放的舒血管活性物质包括前列环素(PG I2)、内皮依赖性舒张因子(EDR F)、内皮依赖性超极化因子(EDH F)等。现已知EDR F的化学本质是一氧化氮(NO),在V EC有产生NO的体系,左旋精氨酸(L2A rg)是合成NO的前体物,乙酰胆碱(A ch)与内皮细胞膜上的M2受体结合后,使内皮细胞中的三磷酸肌醇(IP3)浓度升高,进而升高[Ca2+],在钙调素的辅助作用下,激活细胞内的一氧化氮合成酶(NO S),NO S在辅酶存在时便可使L2 A rg转变为对羟基2L2A rg,后者与氧发生反应生成等克分子量的NO和L2胍氨酸[2]。NO通过激活鸟苷酸环化酶使细胞内cG M P增高,而产生一系列生物效应:松驰血管平滑肌、维持血管舒张状态;抑制血小板的粘附聚集;抑制白细胞粘附分子CD11, CD18的活性或其表达;抑制平滑肌细胞分裂增殖;减少胶原纤维、弹力纤维的产生;清除自由基,抑制脂质过氧化反应。内皮源性一氧化氮(EDNO)对血管内皮功能完整性有重要作用,包括3个方面:①保持血管内皮依赖性舒张活性;②维持血管内膜的无血栓形成表面;③维持血管内膜的非增殖状态[3]。 2 血管内皮细胞与心血管疾病 2.1 血管内皮细胞与高血压 在原发性高血压患者及实验性高血压动物,均发现血浆ET水平较正常人及动物高,其升高水平与高血压程度相关。高血压时血管的结构和功能发生明显改变,阻力血管的基础的和A ch诱发的EDR F合成与释放均显著降低[4]。 2.2 血管内皮细胞与冠心病 L efer等(1991)在动物实验中发现,心肌缺血再灌注后V EC的功能可发生紊乱,特征是舒血管物质释放减少,缩血管物质作用增强。在缺血造成的V EC损伤中,白细胞参与并释放损害V EC的物质,这在冠状循环中尤其明显。 国内外已有大量的临床及实验资料表明,急性心肌梗死时,血浆中内皮素水平明显升高,可升至原来的3倍～5倍,梗死面积越大,内皮素水平越高,而且,这种变化在心肌梗死的早期就已发生。 2.3 血管内皮细胞与心力衰竭 Katz等研究了充血性心力衰竭的动物模型及原发性扩张型心肌病患者冠状循环中内皮舒张功能的变化,发现心力衰竭时患者血管内皮结构及功能均受到损害,自分泌舒血管物质能力下降,相反,缩血管物质的释放增多[5]。心力衰竭时内皮功能障碍的可能机制包括:①肿瘤坏死因子(TN F)增多减少内皮NO S的合成; ②血管紧张素转换酶(A CE)活性增高加快缓激肽降解;③氧自由基增多减弱EDR F NO的活性;④血流量的减少使内皮NO S的表达减弱;⑤内皮依赖性血管收缩物质如环氧合酶依赖因子的增多减弱了NO的扩血管作用;⑥内皮受体信号传导途径受损[6]。长期的A CE抑制剂治疗可以改善心力衰竭已 ? 6 2 1 ?心脏杂志(Ch in H eart J)2000,12(2)