CNP -hEGFR mice GFAP + cells display a morphology of protoplasmatic astrocytes (Sup.Fig. 1d1)16. GFAP and Nestin were co-expressed in the WT SVZ, while the percentage of GFAP +Nestin + cells was reduced in CNP -hEGFR mice (Fig. 1a,b).In SVZ cells from CNP -hEGFR mice, Lex +Nestin +GFAP + NSCs were reduced, compared to WT (Fig. 1a,b). This was not due to cell death (0.6+/?0.02 and 0.5+/?0.06% of Caspase3+ cells/106 um 3, WT and CNP -hEGFR, respectively). Ultrastructural analysis 17confirmed fewer NSCs and more neuroblasts in CNP -hEGFR mice compared to WT (Sup.Fig. 3).We used LeX antibodies 18,19 to FACS-purify LeX +CNP -EGFP neg SVZ NSCs from CNP -EGFP and CNP -EGFP/CNP -hEGFR mice (Sup.Fig. 4). LeX +CNP -EGFP neg cells were GFAP + (data not shown). The number of LeX +CNP -EGFP neg NSCs was reduced in the CNP -hEGFR mouse, whereas LeX +CNP -EGFP + NPCs were increased (Sup.Fig. 4).LeX +CNP -EGFP neg NSCs from CNP -hEGFR mice displayed a reduction in LeX +neurosphere 19,20 number and size, compared to WT cells (Fig. 1c,d). Conversely, CNP -EGFP +LeX + NPC proliferation was increased (not shown).In GFAP -GFP/CNP -hEGFR mice, GFAP -GFP + cells with radial glia-like morphology were almost absent in the SVZ (Sup.Fig. 5b). Conversely, in GFAP -GFP mice, GFP + cells displayed radial glia-like cell morphology throughout the SVZ (Sup.Fig. 5a). In FACS-purified cells from GFAP -GFP and GFAP -GFP/CNP -hEGFR mice, enhanced EGFR signaling reduced the number of LeX +GFAP -GFP + NSCs, whereas the number of LeX +GFAP -GFP neg NPCs was increased (Sup.Fig. 5c,d). NSCs from GFAP -GFP/CNP -hEGFR mice displayed reduced neurosphere number and size compared to cells from GFAP -GFP mice (Sup.Fig. 5c,d), however proliferation of LeX +GFAP -GFP neg NPCs was increased (not shown). These results show that enhanced EGFR signaling and expansion of SVZ CNP -expressing progenitors 14,15 reduces the number, proliferation, and self-renewal of GFAP -expressing NSCs.
Notch 10,21,22, BMP 23,24 and Shh 25 regulate NSC properties in the SVZ. Enhanced EGFR
signaling upregulated genes involved in neurogenesis [Sup. Table 1 (GEO access number
GSE21913) and Fig. 2a], whereas Notch signaling elements were downregulated. Shh and
BMP pathways were not modified (data not shown). In WT and CNP -hEGFR SVZ Notch1was mainly detected in NSCs 22,26, whereas Dll1 and Jagged1 were detected in NPCs and in
neuroblasts (Sup.Fig. 6a–f; see also Fig. 4a–d). Dll -EGFP electroporation demonstrated expression in EGFR +NG2+ and in Dcx + cells, but not in GFAP +
cells (Sup.Fig. 6g–j).Conversely, Hes1 activity was mainly observed in GFAP + cells (Sup.Fig. 6k–m; see also
Fig. 4a–d). Notch1, NICD, Dll1, RBPJK and Hes1 proteins were decreased by 60 ± 3, 63 ±
4, 20 ± 2, 30 ± 2.5 and 40 ± 3% upon enhanced EGFR function, respectively (n= 3–4 each;
Fig. 2a). Conversely, Numb was increased by 45 ± 3% (n=3). In the SVZ of the Wa2
mouse 14, NPC number and proliferation were reduced27, and Notch1, NICD, RBPJK and
Hes1 were upregulated, as compared to WT (Fig. 2b).
EGF infusion into the lateral ventricle of GFAP -GFP mice increased the number of BrdU +
cells in the SVZ, downregulated Notch signaling and expanded EGFR-expressing NPCs
(Sup.Fig. 7a-e). Proliferation and self-renewal of NSCs from EGF-infused SVZ were
reduced compared to controls (Sup.Fig. 7f,g).
To rescue the NSC phenotype in the postnatal CNP -hEGFR mice, we overexpressed the
constitutively active form of Notch1 (Notch1 intracellular domain; NICD) in CNP -hEGFR
SVZ cells (Sup.Fig. 8a). Neurosphere numbers and size were greater in NICD - than in
mock-transfected cells (Sup.Fig. 8b). NICD electroporation in SVZ cells of CNP -hEGFR /
GFAP -GFP mice partially rescued the radial glia-like morphology of GFAP -expressing cells
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(Fig. 2c,d; see also Fig. 4a1–a3 and b1–b3), and increased neurosphere number and size (Fig. 2e,f). After adenovirus-mediated transduction of NICD (Fig. 2g) in adult WT and CNP -hEGFR mice, NICD levels were higher (30 ± 3%) in NICD -LacZ- (ad-NICD ) than in LacZ adenovirus (Ad-LacZ)-infected SVZs, whereas Numb levels were reduced (55 ± 4%)(Fig. 2h). NICD transduction rescued NSC properties in the SVZ of CNP -hEGFR/GFAP -GFP mice (Fig. 2h,i).We infused cytosine-β-D-arabinofuranoside (Ara-C) into the LV to deplete dividing NPCs 9.In saline-perfused adult mice, BrdU + cell number was significantly higher in CNP-hEGFR mice (24.1+/?2.1 cells/106 μm 3) compared to WT (12.4 +/?0.5 cells/106 μm 3) (Sup.Fig.9a,b; t -test p<0.008), whereas NSC number was reduced (Sup.Fig. 9a,b, and Fig. 3a; t -test p<0.005). Ara-C increased GFAP + cell number in the SVZ of adult CNP -hEGFR mice (Sup.Fig. 9c,d and Fig. 3a). We observed higher levels of Notch1 in GFAP + NSCs of Ara-C-treated CNP -hEGFR and WT mice, compared with saline-infused mice (not shown). In CNP -hEGFR mice, Hes1 and RBPjk mRNAs and Notch1 protein levels were higher in Ara-C-treated SVZ tissues than in saline controls (Fig. 3b), whereas Numb mRNA and protein were reduced (Fig. 3b). Cell proliferation and self-renewal were enhanced in neurospheres from Ara-C-treated tissue, compared with saline (Fig. 3c). Altogether, these data indicate that NPCs regulate NSC proliferation and self-renewal in vivo.To demonstrate that contact with NPCs regulates NSC proliferation and self-renewal through Notch, we co-cultured confluent SVZ NPCs from WT or CNP -hEGFR mice with NSCs FACS-purified from GFAP -GFP mice (Sup.Fig. 10). GFAP -GFP + NSCs were then FACS-purified from these co-cultures. Notch signaling was downregulated in NSCs co-cultured with CNP -hEGFR NPCs, and cell proliferation and self-renewal were reduced (Fig.3d and Sup.Fig. 10c). NICD transduction partially rescued this phenotype (Fig. 3d). In NSCs FACS-purified from Wa2 NPC/WT NSC co-cultures Notch signaling and neurosphere formation were enhanced, as compared to WT NPC/WT NSC in NSCs (Fig. 3e and Sup.Fig.
10f). These results confirm that EGFR regulates Notch through a non-cell-autonomous
mechanism.
We determined whether EGFR regulates the Notch pathway and investigated the molecular
mechanism. In transfected SVZ cells, CBF-1 and Hes1 promoter activity was higher in WT
than in CNP -hEGFR cells, even after NICD co-transfection (Sup.Fig. 11a). In WT cells,
Hes1 activity was enhanced by NICD and reduced after hEGFR co-transfection (Sup.Fig.
11b). hEGFR co-transfected with different Notch constructs (Sup.Fig. 11c) reduced Hes1
activation (Sup.Fig. 11d), and the EGFR inhibitor PD16839315 restored Hes1 activity
(Sup.Fig. 11e). CNP -hEGFR cells plated on WT cells transfected with Hes1-luciferase
suppressed basal Hes1 activity; this effect was reversed by PD168393 (Sup.Fig. 11f).
Finally, Hes1 promoter activity was higher in Wa2 SVZ cells than in WT cells (Sup.Fig.
11g).
We electroporated Notch target constructs (Sup.Fig. 12) to identify SVZ Notch-responsive
cells and to elucidate the mechanism of Notch regulation. Hes1 activity was observed in
NSCs (Fig. 4a-d and Sup.Fig. 12m,n), and was found in a larger percentage of GFAP -GFP +
cells (47.8+/?3.6 cells/μm3) than in GFAP -GFP +/CNP -hEGFR + cells (23.6+/?4.6 cells/
μm3, p<0.01). Similar results were observed with CBFRE -GFP and Hes5-GFP (Sup.Fig.
12a–l; o–r). Hes1 activity in NSCs of GFAP -GFP/CNP -hEGFR mice was rescued by NICD
co-electroporation (66.5+/?71 cells/μm3, p<0.01; n=4). In Wa2 mice, a larger percentage of
SVZ NSCs displayed Hes1 and CBFRE activity, compared to WT (Sup.Fig. 13a–c, and not
shown). shRNA-mediated knockdown of hEGFR in CNP -hEGFR mice in vivo caused
upregulation of Notch1 and Hes1 (Sup.Fig. 13d,e). Consistent with these results, Dll-EGFP
electroporation revealed a larger percentage of Dll -EGFP + cells in the SVZ of the WT
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mouse, compared to CNP -hEGFR mouse (Sup.Fig. 14). Altogether, our findings
demonstrate that EGFR is an upstream regulator of Notch through a non-autonomous
cellular mechanism.
Numb interacts with E3 ubiquitin ligases to regulate Notch receptor degradation 28. Changes
in Notch signaling levels directly regulate Numb expression 29. Figure 4e shows that NICD
overexpression reduced Numb in SVZ cells, whereas EGFR overexpression upregulated
Numb, and reduced Notch1 and NICD levels. The Notch inhibitor DAPT upregulated Numb
(Fig. 4f).
We determined the extent of Numb/Notch1 interaction in the SVZ by immunoprecpitation
assays. Higher levels of Notch1 associated with Numb in CNP -hEGFR samples, compared
to WT (Fig. 4g1). Anti-ubiquitin immunoprecipitation followed by anti-Notch1
immunoblotting demonstrated higher levels of Notch ubiquitination in CNP -hEGFR mice
(Fig. 4g2). Stereotaxic injection in WT and CNP -hEGFR mice with either Ad-LacZ or
NICD -LacZ adenovirus showed that both Numb/Notch interaction and Notch1
ubiquitination were reduced after NICD transduction (Fig. 4h).
To demonstrate that EGFR signaling promotes Notch ubiquitination via Numb, we
performed gain- and loss-of-function experiments in SVZ cultures. After EGFR transfection,
we observed increased Numb levels and reduced NICD expression, compared to the mock
construct (Fig. 4i). PD168393 reversed this effect (Figure 4i). EGFR overexpression in WT
cells promoted Numb and Notch association and enhanced Notch1 ubiquitination (Fig. 4j).
siRNA-mediated EGFR knock down in CNP -hEGFR SVZ cultures reduced Numb and
Notch1 interaction (Fig. 4k1), and decreased Notch1 ubiquitination (Fig. 4k2).
To confirm the role of EGFR and Numb in inhibiting Notch, we analyzed Hes1 promoter
activity by co-transfection in WT SVZ cells with a Hes1-luciferase reporter construct, along
with hEGFR and Numb expression vectors. Hes1 activity was activated only by the NICD
construct (Sup.Fig. 11h). However, Numb blocked NICD and reduced Hes1 activity
(Supplementary Fig. 11h). Hes1 activity was further reduced by co-transfection with NICD ,
Numb , and hEGFR (Sup.Fig. 11h). Finally, siRNA mediated knockdown of Numb in vivo
caused upregulation of Notch1 and Hes1 activity (Fig. 4l–o). Scrambled siRNA
electroporation in vivo showed that Hes1 activity was present in a small percentage of
GFAP + and Nestin + NSCs (Fig. 4n; 3.9+/?0.51 cells/μm3), however after Numb knock
down Hes1 activity was present in a larger percentage NSCs (Fig. 4o; 7.1+/?0.56 cells/μm3,
p<0.01; n=4). Together, these results demonstrate that EGFR signaling reduces Notch
activation through Numb-dependent Notch1 ubiquitination.
Our results point to an interaction between two signaling pathways -EGFR and Notch - that
play fundamental and selective roles in the maintenance of NSCs and NPCs in the SVZ
niche. This interplay occurs through the direct interaction between NPCs and NSCs,
demonstrating the existence of a cellular homeostatic mechanism that involves two specific
molecular pathways. Our study also proves that altering particular signaling mechanisms in
selective cell types of the SVZ can cause profound changes in the overall cell composition
of this neurogenic region of the adult brain. Defining interactions and homeostatic
mechanisms that occur between different types of SVZ cells under normal conditions
provides crucial information on possible alterations of specific signaling pathways that
might occur under pathological conditions or after brain injury.METHODS SUMMARY
Generation/genotyping of CNP -hEGFR, hGFAP -GFP and Wa2 mice was performed as
described14,15. Wild-type C57/Bl6 and FVB/N mice were used as controls. Histology,
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immunohistochemistry and EM studies were performed on fresh floating or vibratome
sections as described previously19,20. Immunohistochemestry and confocal imaging were
used to characterize the SVZ cell composition of the postnatal and adult brain. Image
analysis, three-dimensional rendering, and cell counting were done in Photoshop (Adobe)
and ImageJ software. NSCs and NPCs were isolated and characterized by FACS sorting
using CD15, GFAP, EGFR and NG2 antibodies. Cell proliferation, self-renewal and
biochemical analysis were performed from FACS-purified cultured cells and SVZ tissue. To
compare SVZ expression profiles of genes involved in NSC development/neurogenesis in
CNP -hEGFR and WT mice, we performed gene array (SuperArray, Bethesda, MD)
expression on spotted cDNA fragments encoding 250 mouse genes. To monitor Notch-
signaling pathway in the postnatal SVZ of the CNP- and WT mice, mouse brains were
electroporated using the ECM 830 BTX electroporator (Harvard Apparatus, Holliston, MA).
Each electroporation result was reproduced in multiple brains derived from at least three
separate litters. SVZ NPC cell depletion using Ara-C (2%, Sigma) in vehicle (0.9% saline)
or vehicle alone was performed as previously described 3. EGF (100nm/ul, Upstate) in
vehicle (0.9% saline), or vehicle alonewas infused into the LV of adult GFAP -GFP and WT
mice (infusion coordinates: anterioposterior, 0; lateral, 1.1; dorsoventral, 1.5 mm medial to
lateral relative to bregma) for 5 days using micro-osmotic pumps (Alzet, model 1007).
Brains were then processed for FACS-purification, cell culture or histology. At least 3
different brains for each strain and each experimental condition were analyzed and counted.
Cell counting was performed blindly and tissue sections were matched across samples. The
analysis of the SVZ was performed at different anterior-posterior and dorso-ventral levels of
the lateral ventricle. Statistical analysis was performed by unpaired t-test.METHODS Animals Details on the generation and characterization of CNP -hEGFR transgenic mice have been
previously reported 30,15. Genotyping of the mice was performed by PCR 15. Robust hEGFR
expression was detected in total brain and spinal cord lysates from adult brain by using
monoclonal anti-human EGFR antibody to probe Western blots after immunoprecipitation
with a polyclonal anti-EGFR antibody 14,15,30. Consistent with the idea that the CNP
promoter drives expression in OLs, hEGFR expression was detected in OL lineage cells of
the SVZ, white matter and cerebral cortex in P8-P90 CNP-hEGFR mice, and in NG2+
progenitor cells of the SVZ 15. Transgenic mice were backcrossed >4 generations onto
C57BL/6. In the CNP -hEGFR mouse strain, the CNP promoter drives expression in NPCs
and in the entire oligodendrocyte lineage; hEGFR expression was detected at both P8 and
P90 in NG2+ progenitors of the SVZ, and in NG2+ progenitors and oligodendrocytes of the
white and gray matter 14,15. The mouse strain expressing the hGFAP -EGFP (kind gift of F.
Kirchhoff, Max Planck Institute of Experimental Medicine, Goettingen, Germany) was
previously characterized31. The EGFR-mutant mouse (waved-2 mutation; Wa2)32 was
obtained from Jackson Labs (Bar Harbor, ME). All animalprocedures were performed
according to the Institutional Animal Care and Use Committee of Children’s National
Medical Center and the National Institutes of Health “Guide for the Care and Use of
Laboratory Animals.”
Immunohistochemistry and antibodies
Freshly cut, floating tissue sections (20–40μm) from P8 and P90 mice were prepared as
previously described14,15. Primary antibody dilutions were: 0.5μg/ml for the specific anti-
hEGFR (Biofluids, Caramillo, CA); 1:500 for anti-BrdU (Accurate, Westbury, NY), anti-
NG2 antibody (Chemicon, Temecula, CA), monoclonal anti-GFAP (mouse monoclonal,
Sigma Aldrich, St. Louis, MO), polyclonal anti-GFAP (rabbit polyclonal, Covance,
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Berkeley, CA), anti-S100β (DAKO, Denmark; rabbit anti-human clone A5110), and anti-
Nestin (Chemicon); 1:50 for anti-LeX (MMA clone, BD Biosciences, San Jose, CA), anti-
full length Notch-1, Jagged1, Delta like-1 (Dll) and anti-NICD (from Developmental Studies
Hybridoma Bank, Iowa, IA).
BrdU administration
The BrdU labeling protocol was performed as previously described33,19. The number of
proliferating progenitor cells in the subependyma of the lateral ventricle was determined
following short-term (2h), or long-term (30d) retention. Mice were injected intraperitoneally
with a single dose of BrdU (50 μg/g body weight) every 3h for five injections and were
sacrificed either 1h or 30d after the final injection.
Conventional transmission electron microscopy
Wild type and CNP -EGFR mice (P8, n=6 for each phenotype; adult, n=4 for each
phenotype) were perfused through the heart with a mixture of 3% paraformaldehyde and
1.25% glutaraldehyde in 0.1M phosphate buffer (PB). Brains were removed and postfixed
overnight with the same fixative. Brains were sectioned with a vibratome and stored in 0.1M
PB. Before postfixation for 1 hour with 1% osmium tetroxide, slices were washed in 0.1M
cacodylate buffer pH7.2. After, brain slices were dehydrated through a series of ethanol
solutions (50%, 70%, 85%, 95% and 100%), infiltrated with epoxy resins and flat
embedded. Sections with the lateral ventricle and the SVZ were trimmed and mounted on
blocks and cut with an ultramicrotome. Ultrathin sections (70–80 nm in thickness) were
counterstained with uranyl acetate and lead citrate and analyzed with a TECNAI G2 Spirit
Biotwin TEM (FEI, Hillsboro, OR, USA). The images were captured with an AMT XR40 4
megapixel side mounted CCD camera (Danvers, MA, USA) using the electron micrograph
montage option. Image processing was performed with Adobe Photoshop using only the
brightness and contrast commands. The identification of cell types in the lateral wall of the
lateral ventricle and SVZ was performed on electron micrographs following well-described
ultrastructural criteria by Doetsch et al. (1997). Cells were false colored and were counted
manually.
Microarray and PCR analysis
GEArray ? expression array systems (cat #OMM-404MM and #OMM-404MM,
SuperArray, Bethesda, MD) consisting of spotted cDNA fragments encoding 250 mouse
genes involved in NSC development/neurogenesis and in Nocth signaling, as well as control
sequences (PUC18, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), peptidylpropyl
isomerase A (Ppia), and b-actin) were employed to compare gene expression between WT
and CNP -hEGFR SVZ tissue. Total RNA was isolated with theTrizol method (Invitrogen)
and further processed for microarray hybridization according to the manufacturer’s
instructions. The arrays were visualized by autoradiography and hybridization signals were
scanned and analyzed for density in GEArray Expression Analysis Suite 2.0. The
normalized value for each gene was calculated by dividing the value of each gene by the
average value of the housekeeping genes GAPDH, Ppia, and β-actin.
For RT-PCR, RNA was isolated from P8 and P90 SVZ tissue or from FACS-purified SVZ
cells (WT and CNP-hEGFR mice), using Trizol (Invitrogen). RNA (1μg) from each sample
was reverse-transcribed using the SuperScript ? First-Strand cDNA Synthesis kit
(Invitrogen). The mouse gene-specific primers were obtained from Integrated DNA
Technologies, Inc. (Coralville, IA). Primer sequences for PCR analysis are found in
Supplementary Experimental Procedures. Genes were amplified by denaturation at 94°C for
1 min, annealing at 60°C for 1 min, and extension at 72°C for 1 min for 28 cycles. PCR NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
products were resolved by 1.2% agarose gel electrophoresis and visualized by ethidium
bromide staining.
In vivo electroporation For gene transfer into postnatal SVZ cells, GFAP -GFP and GFAP -GFP/CNP -hEGFR P2-P3pups were injected with 1–2μg of pFLAG-NICD (gift from Dr. Raphael Kopan),shhumanEGFR (GeneCopoeia, Cat #HSH004605-LvH1, access number NM_005228.3;HSHH004605 actcactctccataaatgc; HSH004605-2 cgtcagcctgaacataaca; HSH004605-3gaccagacaactgtatcca; HSH004605-4 ccgtcgctatcaaggaatt), CAG -GFP and Hes1-dsRED and Hes1-GFP (gift from Dr. Cepko L. Constance)34 CBFRE-EGFP (gift from Dr. Nicholas Gaiano)22, Dll -GFP (gift from Richard Grosschedl)35 and Hes5-GFP (gift from R.Kageyama)36 DNA into the LV, followed by electroporation (100V/50ms, five times at 950ms intervals; the angle of the paddles was adjusted to 20–40 degrees) using an ECM 830BTX electroporator (Harvard Apparatus, Holliston, MA).In vivo viral labeling SVZ cells were infected using a Notch Intracellular domain (Ad-NICD) or control (Ad-LacZ) construct Adenovirus (gift of Dr. Igor Prudovsky) by direct injection into the LV.Adenovirus production and titer determination were previously described 37. P90 WT and CNP -hEGFR mice were injected with the Adenovirus stock (2μl; titer of 2–4 × 106 cfu/ml).Injections were performed stereotaxically at the following coordinates (anterioposterior relative to bregma, mediolateral, and dorsoventral from surface of the brain) for epl-SVZ (0,1.8, 3.0mm). Brains were processed for histology at 2, 7, 14 and 28 days after infection, and sections were immunostained for NICD, GFAP, S100b, NG2 and BrdU antibodies.FACS sorting and cell culture FACS-purification of LeX +NG2?/EGFP neg (NSCs) in the CNP -EGFP mouse or GFAP-
GFP +LeX +NG2neg (NSCs) in the GFAP -GFP mouse has been previously described 19.
Tissue from P8 CNP-hEGFR, CNP -EGFP/CNP -hEGFR, GFAP -GFP or CNP -hEGFR/
GFAP -GFP mice was dissociated into single cell suspensions, followed by immunostaining
for NG2 and LeX (MMA clone)19. SVZ neural stem cells and NPCs (after EGF or saline
infusion, or 5–7days after focal demyelination of the corpus callosum) were FACS-purified
using anti-LeX antibodies to purified LeX +/GFAP -GFP + (NSCs) or GFAP -GFP neg LeX +
(NPCs) cells from the hGFAP -GFP mouse. Tissue was dissociated into single cell
suspensions, followed by immunostaining for NG2 and LeX (MMA clone)19. To FACS-
purify NSCs, antibodies were used in combination with appropriate R-PE- and PE-Cy5.5-
conjugated secondary antibodies (Caltag, Burlingame, CA). Cell suspensions were analyzed
for light forward and side scatter using a FACSAria instrument (BD, Biosciences, Franklin
Lakes, NJ).
Cultures of FACS-purified cells have been previously described 19. FACS-purified NSCs
were seeded at a density of 10 viable cells/μl on uncoated 24-well plates (BD Falcon,
Franklin Lakes, NJ), and grown in SCM for 6 days in vitro (DIV) with daily addition of
20ng/ml EGF and 10ng/ml bFGF (Upstate, Charlottesville, VA)19,20. Primary neurosphere
colonies were subcloned to assay NSC self-renewal by mechanical dissociation and replated
at a density of 10cells/μl on uncoated 24-well plates. It is important to note that in
Supplementary Figure 6 neurosphere analysis was performed starting with the 2nd passage.
This was done to overcome the experimental limitations due to low number of cells
recovered after FACS.
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SVZ micro-dissection
SVZ areas were micro-dissected from 300μm-thick brain coronal sections. Single cell were
dissociated19,38 on coated coverslips in 24-well plates (BDFalcon, Franklin Lakes, NJ).
Coverslips were processed for immunocytochemistry 2h after plating.
Western blots and immunoprecipitation
SVZ tissue (WT and CNP -hEGFR mice) was micro-dissected from 300μm-thick coronal
sections of P8 and P90 brains, and used for protein extraction using lysis buffer (50mM
Tris-HCl, pH7.5, 1mM EDTA, 1mM EGTA, 1mM sodium orthovanadate, 50 mM sodium
fluoride, 0.1% 2-mercaptoethanol, 1% triton X-100, plus proteases inhibitor cocktail;
SIGMA). Protein samples (20μg) were separated on GENE Mate express Gels 420% (ISC
BioExpress, Kaysville, UT) and transferred to PVDF membranes (Millipore, Bedford MA).
Numb, Notch1, NICD, Dll1, Hes1, RBPjk, ubiquitin, and actin proteins, were detected using
an enhanced chemiluminescence substrate mixture (ECL Plus, Amersham,
Buckinghamshire, UK). Selective primary antibodies from Santa Cruz Biotechnologies
(Caramillo CA) were used at 1μg/ml. Antibodies were used in combination with a secondary
horseradish peroxidase-conjugate (Santa Cruz Biotechnologies).
For immunoprecipitation, SVZ tissue extracts from WT and CNP-hEGFR mice were
prepared in RIPA buffer containing. Aliquots (200μg protein) were incubated overnight with
antibodies against ubiquitin (Santa Cruz Biotechnology), Numb or Notch1 and 15μl of
Agarose A (Santa Cruz Biotechnology). Immunocomplexes bound to agarose A were
collected by centrifugation and washed twice in 500μl RIPA buffer containing inhibitors.
Precipitated proteins were analyzed by immunoblotting. Bands were detected using HRP
and developed with a chemiluminescent substrate (ECL, Amersham).
Ara-C and EGF infusion
Ara-C (2%, Sigma) in vehicle (0.9% saline) or vehicle alonewere infused onto the surface of
the brain of adult mice (coordinates: anterioposterior, 0, lateral, 1.1, dorsoventral, 1.5mm
medial to lateral relative to bregma) for 6 days with micro-osmotic pumps (Alzet, model
1007) as previously described by Doetsch et al. (1999). EGF (100nm/ul, Upstate) in vehicle
(0.9% saline), or vehicle alone was infused into the LV in adult GFAP -GFP and WT mice
(infusion coordinates: anterioposterior, 0; lateral, 1.1; dorsoventral, 1.5 mm medial to lateral
relative to bregma) for 5 days using micro-osmotic pumps (Alzet, model 1007). Mice were
then processed for FACS-purification, cell culture or histology.
Plasmids, siRNAs, cell transfection and luciferase assays
SVZ tissue was dissected from 300μm-thick brain sections prepared from P8 WT and CNP-
hEGFR brains and processed for cell culture 19. After SVZ dissection and single cell
dissociation, cells were plated in 12-well cell culture dishes at a density of 50 cells/μl for 24
hours. At the time of transfection, cell cultures were approximately 60% confluent. Cell
transfections were performed using the NeuroPORTER Transfection reagent (Genlantis, San
Diego, CA) following manufacturer’s instructions. The cell permeable, irreversible EGFR
blocker (PD168393, Calbiochem, La Jolla, CA), was used 24hrs after cell transfection. Cells
were pre-incubated with PD168393 (PD) for 4hrs at 37°C in 5% CO 2 and then medium was
changed to fresh SCM.
A commercially available siRNA directed toward the hEGFR was purchased from
Dharmacon (SMARTpool Cat. No. L-003114-00, Locus NM_201283; sequence
J-003114-10, CAAAGUGUGUAACGGAAUA; J-003114-11,
CCAUAAAUGCUACGAAUAU, J-003114-12, GUAACAAGCUCACGCAGUU,
J-003114-13, CAGAGGAUGUUCAAUAACU. and siRNA directed toward the mNumb
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(Santa Cruz Biotechnologies Inc. Cat. No. sc-42147; Locus NM_010949, 110
GUAGCUUCCCAGUUAAGUAtt, 412 CGAUGGAUCUGUCAUUGUUtt, 884
CCCUACGCAUCAAUGAGUUtt). siRNA transfection produced selective knockdown of
the hEGFR and mNumb. Briefly, 2μl of 20pM of siRNA solution and 12μl of the
transfection reagent were incubated in 100μl of SCM for 20 minutes, in order to facilitate
complex formation. The siRNA transfection mix was added to the SVZ cells cultured in
2.5% FBS. Controls consisted of non-specific siRNA. SVZ cells were transfected for 7
hours at 37°C, washed with Hanks’ buffer and cultured in SCM 2.5% FBS for an additional
24 hours. The medium was then changed to basal SCM (20 ng/ml EGF and 10 ng/ml FGF).
After 24 or 48 hours, cells were collected and processed for RNA or protein extraction.
Transient transfections and Luciferase assays were performed in 60% confluent SVZ cell
cultures (CNP -hEGFR and WT) using NeuroPORTER as described above, and 1.5μg of
PGL3 basic, wt phEGFR (Millipore), pCBF1-Luciferase reporter plasmid (gift of Dr.
Gabriel Corfas, Harvard Medical School) or Hes1 Luciferase reporter plasmid, and Dll
expression plasmid (gift of Dr. Alanis Israel, Unite de Biologie Moleculaire de l’Expression
Genique, Centre National de la Recherche Scientifique) reporter plasmids and 0.3μg of
expression vectors. Notch expression vectors used were a kind gift of Dr. Raphael Kopan
(Department of Molecular Biology and Pharmacology, Washington University). Luciferase
assays were performed 48hr after transfection using the Dual Assay Luciferase kit
(Promega). Cotransfected TK–renilla Luciferase was used to normalize samples for
transfection efficiency and for sample handling. Cells were lysed, and luciferase activity was
measured following the protocol recommended by the manufacturer.
Neural progenitor-neural stem cell co-cultures
NPCs were acutely dissociated from P8 CNP -hEGFR and WT SVZ mice and then processed
for purification using anti-prominin-1 microbeads antibodies (Miltenyi Biotec, Auburn CA)
following manufacturer recommendations. Purified NPCs were cultured at high density in a
monolayer in the presence of EGF and bFGF, as described above. After 48 hrs, FACS-
purified WT GFAP -GFP +LeX + cells were plated on top of NPCs for 5 days in EGF- and
bFGF-containing medium. Finally, GFAP -GFP + cells were purified from the co-culture by
FACS and assayed in neurosphere cultures to determine proliferation and self-renewal.
FACS-purified GFAP -GFP + cells were seeded at a density of 5 viable cells/μl on uncoated
12mm-well plates (BDFalcon, Franklin Lakes, NJ), and maintained in SCM for 6DIV with
daily addition of EGF and bFGF. Primary neurosphere colonies were subcloned by
mechanical dissociation in SCM with EGF and bFGF. Cells were re-plated at a density of 5
cells/μl on uncoated 24-well plates. Stem cell self-renewal was assessed after further 6 DIV.
Microscopy and cell counting
A Bio-Rad MRC 1024 confocal laser-scanning microscope (Hercules, CA) equipped with a
krypton-argon laser and an Olympus IX-70 inverted microscope (Melville, NY) was used to
image localization of FITC (488nm laser line excitation; 522/35 emission filter), texas red
(568nm excitation; 605/32 emission filter) of Cy5 (647 excitation; 680/32 emission filter).
Optical sections (Z=0.5μm) of confocal epifluorescence images were acquire sequentially
using a 40x oil objective (Number or aperture, NA=1.35), or a 60x oil objective (NA=1.40)
with Bio-Rad LaserSharp v3.2 software. ImageJ NIH, software was subsequently used to
merge images. Merge images were processed in Photoshop 7.0 with minimal manipulations
of contrast. For cell counting, cells were counted in the SVZ the postnatal day 8 (P8) and
adult mice P90. At least 3 different brains for each strain and each experimental condition
were analyzed and counted. Cell counting was performed blindly and tissue sections were
matched across samples. The analysis of the SVZ was performed at different anterior-
posterior and dorso-ventral levels of the lateral ventricle. An average of 15–20 sections was NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
quantifiedusing unbiased stereological morphometric analysis for the SVZ to obtain an
estimate of the total number of positive cells. Then, percentages of cells expressing different
antigens were estimated by scoring the number of cells double-labeled with the marker in
question. In acute SVZ dissociated cells, 3 coverslips and 8–10 microscopic fields/coverslip
were counted from 3 separate cultures. Statistical analysis was performed by unpaired t-test.Supplementary Material Refer to Web version on PubMed Central for supplementary material.Acknowledgments We thank Dr. Nancy Ratner (Cincinnati Children’s Hospital Research Foundation, Cincinnati, OH) for the CNP -hEGFR mice. We are particularly thankful to Teresa Hawley (George Washington University) for technical advice in all FACS sorting experiments. We are grateful to Drs. Josh Corbin and Tarik Haydar for critically reading this manuscript, and to all our colleagues at the Center for Neuroscience Research for discussion and support. We are particularly thankful to Dr. Nicholas Gaiano for discussion, for providing reagents and for his continuous and generous advice on this project. We thank Drs. Gabriel Corfas (Harvard Medical School, Boston, MA), Alanis Israel (Unite de Biologie Moleculaire de l’Expression Genique, Centre National de la Recherche Scientifique, Paris,France), Constance L. Cepko (Department of Genetics, Harvard Medical School, Boston, Massachusetts), Raphael Kopan (Department of Molecular Biology and Pharmacology, Washington University, St. Louis), Richard Grosschedl (Max Planck Institute of Immunobiology, Department of Cellular and Molecular Immunology,Freiburg, Germany), Nicholas Gaiano (Institute for Cell Engineering, Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD), and Ryoichiro Kageyama (Institute for Virus Research, Kyoto University, Kyoto, Japan) for the gift of CBF-1, Hes1-GFP, Hes1-dsRED, Notch constructs, Dll -GFP, CBFRE -EGFP and Hes5-GFP respectively. This work was supported by NIH R01NS045702 and R01NS056427 (V.G.),K99NS057944 (A.A.), ROO NS057944-03 (A.A.) and by NIH IDDRC P30HD40677 (V.G.). Electron microscopy was performed at the University of Connecticut, Department of Physiology and Neurobiology, with funding from NIH R01DC006881 to M.E.R., and from NSF DBI-0420580 for funds to purchase the Tecnai 12 Biotwin electron microscope.References
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Figure 1. EGFR overexpression reduces NSC proliferation and self renewal in the adult mouse SVZ (a) Decreased number of GFAP +BrdU + and GFAP +Nestin + NSCs, and increased number of NG2+ cells in the SVZ of the CNP -hEGFR mouse (*p<0.05). Means ± SEM. (b) Decreased percentage of GFAP +Nestin +LeX + NSCs and increased percentage of GFAP +S100b +
astrocytes in the SVZ of the CNP -hEGFR mouse (**p<0.02). (c) Neurospheres from WT (c1) and CNP -hEGFR (c2) cells. (d) Reduced neurosphere numbers (d1) and size (d2) in cultures from CNP -hEGFR mice (*p<0.02; **p<0.001).
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Figure 2. EGFR overexpression downregulates Notch signaling in the SVZ, and NICD overexpression rescues proliferation and self-renewal of SVZ NSCs (a) Notch signaling is downregulated, but Numb is upregulated in the SVZ of CNP -hEGFR mice. (b) NICD, RBPjk, Hes1 are upregulated in the Wa2 SVZ, and Numb is downregulated. (c-d) High NICD levels were detected after in vivo electroporation by
immunostaining. (e–f) GFAP-GFP + neurosphere number (f1) and size (f2) increased after NICD electroporation. Means (n=3) ± SEM (*p<0.01). Scale bars = 100μm. (g) High b-gal expression levels in the SVZ after viral infection in WT (g1) and CNP -hEGFR (g2)
ventricles. (h) Ad-NICD transduction increased NICD, but reduced Numb expression. (i)Ad-NICD infection increased neurosphere formation in CNP -hEGFR mice, but not in WT.Means (n=3) ± SEM (*p<0.02). Scale bars = 200μm.
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Figure 3. EGFR-expressing NPCs regulate NSC properties through a non-autonomous cellular mechanism (a) Increased BrdU + cells and reduced GFAP +
cells in CNP -hEGFR mice (**p<0.002).AraC: Increased GFAP + cells compared with NaCl in CNP -hEGFR mice (**p<0.002;
*p<0.05). (b1) AraC upregulates Hes1 and RBPjk mRNAs and downregulates Numb . (b2)AraC upregulates NICD protein and downregulates Numb. (c) CNP -hEGFR mice: AraC increases NSC proliferation and self-renewal (*p<0.001). Means (n=3) ± SEM. (d) Reduced proliferation and self-renewal in WT NSCs cultured with CNP -hEGFR NPCs, as compared with WT cells. Ad-NICD overexpression rescued NSC proliferation. Means (n=3) ± SEM (*p<0.02). (e) Higher proliferation and self-renewal of NSCs cultured with NPCs of the Wa2 mouse, as compared with WT cells. Means (n=3) ± SEM (*p<0.05).
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Figure 4. EGFR signaling reduces Notch1 expression through Numb (a–d) Hes1-dsRED-NICD co-electroporation in GFAP -GFP and GFAP -GFP/CNP -hEGFR mice. More Hes1-dsRED +GFAP -GFP + cells are observed in GFAP -GFP mouse. NICD increases GFAP -GFP +Hes1-dsRED + cell number. (e) WT cells infected with: i) LacZ or NICD , or ii) GFP retrovirus (CLE -GFP) or EGFR -GFP. NICD upregulates NICD and
Notch1, but downregulates Numb. EGFR reduces Notch1 and NICD, but increases Numb.(f) DAPT upregulates Numb. (g) Increased Numb/Notch1 immunoprecipitation correlates with enhanced degradation. (h) Ad-NICD reduces Notch1/Numb interaction and Notch1degradation in CNP -hEGFR SVZ. (i) EGFR increases Numb and reduces NICD; PD168393prevents these effects. (j) EGFR increases Numb/Notch immunoprecipitation, and Notch1degradation. (k) hEGFR siRNAs decrease Numb/Notch1 interaction and Notch1 degradation in CNP -hEGFR cells. (l–o) Numb siRNA knockdown enhances Notch signaling in CNP -hEGFR NSCs. Scrab=scrambled siRNA. (l) Numb siRNA downregulates Numb and upregulates Notch1. (m) Numb siRNA decreases Numb/Notch interaction and Notch1degradation. (n–o) Co-electroporation of Scrab or Numb siRNA, and Hes1-dsRED
constructs. Numb siRNA increases the number of Hes1-dsRED +CAG -GFP + cells. Scale bar = 50μm.
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肺癌干细胞的研究现状 发表时间：2011-06-24T10:37:06.280Z 来源：《中国健康月刊（学术版）》2011年第2期供稿作者：梁冬马秀梅[导读] 肺癌已成为世界上发病率和死亡率增长最快，严重危害人类健康和生命的恶性肿瘤。 梁冬马秀梅黑龙江省青年科学基金项目编号QC2009C95 【摘要】肺癌已成为世界上发病率和死亡率增长最快，严重危害人类健康和生命的恶性肿瘤。最近，科学家们提出“肿瘤干细胞”理论，认为肿瘤组织由异质性的细胞群体组成，其中很小部分细胞具有干细胞特性，是恶性肿瘤发生、耐药、复发及转移的根源。因此人们希望从肿瘤干细胞的角度找到根治肺癌的途径。 【关键词】肺癌;干细胞;肿瘤干细胞 【中图分类号】R725【文献标识码】A【文章编号】1005-0515(2011)02-0227-02 目前，肺癌已成为世界上发病率和死亡率增长最快，严重危害人类健康和生命的恶性肿瘤，5年生存率低于15%。科学家们通过总结大量肿瘤细胞和干细胞的生物学相似性后，提出“肿瘤干细胞” (TSC)理论。该理论的提出为肺癌的治疗带来曙光。 1干细胞 干细胞(SC)是指具有自我更新和分化潜能的细胞。干细胞的自我更新和分化在其内在机制和周围环境中的信号调控下处于动态平衡状态,维持了干细胞的数量稳定。一旦干细胞发生基因突变或信号传导途径发生错误,将导致这一平衡被打乱,引起高度协调的干细胞分裂增殖过程失调,导致肿瘤发生。 2肿瘤干细胞 2001年，科学家们提出了TSC理论，认为肿瘤组织由异质性的细胞群体组成，其中很小部分细胞具有干细胞特性，决定肿瘤的发生、侵袭、转移、播散和对各种治疗是否敏感[1~3]。TSC的最早报道见于白血病。Ai-Hajj等发现乳腺癌干细胞,首次证明了在实体瘤中TSC的存在。目前已成功分离并鉴定的实体TSC包括脑肿瘤、结肠癌、前列腺癌、黑色素瘤及胰腺癌等,肺癌TSC的研究也取得很大进展。 3肺癌干细胞 3.1肺癌干细胞的发现 2005年Kim等从大鼠细支气管、肺泡管结合部分离出Sca-1+CD45-Pacam-CD34- 细胞,其有很强的自我更新和分化能力, 称之为支气管肺泡干细胞(BASCs)，并认为BASCs可能是肺腺癌的起源细胞。2006年,黄盛东等发现,A549细胞悬浮培养可形成3种类型的克隆集落,其中Holoelone型克隆体具有干细胞特性。2007年,Summer等从鼠的肺组织中分离出肺内源的间充质干细胞。Ho等发现肺癌SP细胞较非SP细胞具有更强的致瘤性, 表达乳腺癌耐药蛋白(ABCG2)等多种ABC家族膜转运蛋白。Eramo等在人的肺癌组织中发现一群具有自我更新、多向分化能力的CD133+细胞，其具有肿瘤细胞的恶性特征，被命名为肺癌干细胞。 3.2肺癌干细胞的分选方法:肺癌干细胞的分选主要采用三种方法:A.应用细胞表面特异性标记进行分选;B.根据SP表型进行TSC的分选;C.利用干、祖细胞中ALDH的含量较高进行分选。 细胞表面标志物是分选肺癌肿瘤干细胞的关键。Kim等应用流式细胞仪分选出Sca+/CD45-/Pecam-/CD34+的支气管肺泡干细胞，肺腺癌肿瘤干细胞亦存在与BASCs相似的表面标志物。另一表面标志物是CD133。Eramo等发现CD133+细胞在肺癌标本中普遍存在，但含量较低。科学家们还发现，CD133+肺癌细胞可无血清悬浮培养，含血清培养出现分化； CD133+细胞比CD133-细胞更易形成与原发瘤表型一致的肿瘤，更具耐药性。上述研究显示CD133+细胞可能包含了肺癌干细胞，且和肺癌化疗耐药密切相关。但Meng等[19]对肺癌细胞株中的CD133+及CD133-细胞进行对比，发现二者均可形成移植瘤，故CD133+细胞是否代表肺癌干细胞，仍存在争议。 人们发现造血干细胞具有将荧光染料泵出细胞外的特性,即SP特性[23]。目前利用这一特性进行纯化已成为一种常用的分离方法。SP 细胞表面表达ABCG2等ABC家族膜转运蛋白,因此,也可应用ABCG2作为标记分选TSC。利用干、祖细胞中ALDH的含量较高进行分选肺癌干细胞。在造血系统、神经系统的干、祖细胞、乳腺癌中ALDH含量很高。在乳腺癌中发现ALDH1+肿瘤细胞具有干细胞特性，且与乳腺癌不良预后相关。美国学者应用流式细胞仪从人类肺癌细胞系中分选出ALDH1阳性细胞，发现其具备诸多干细胞特征，并表达CD133，并与肺癌患者的预后呈负相关。 3.3肺癌干细胞的鉴定方法:目前研究认为TSC鉴定必须通过一些经典的干细胞实验进行鉴定。自我更新是干细胞的三大重要特性之一,如 CD133+肺癌细胞能够在含生长因子的无血清培养基中以细胞球的方式悬浮生长,这是鉴定其具有自我更新的能力,也是鉴定其是为TSC的重要方法。致瘤能力是鉴定TSC的最重要环节,目前多通过裸鼠致瘤实验进行检验。分化潜能是TSC的重要特征之一，可通过单克隆实验进行检验,以此鉴定其干细胞特性。 4肺癌干细胞的展望 进入21世纪以来，肺癌的治疗已经进入分子时代,对肺癌干细胞的研究越来越受到重视。直接靶定肺癌干细胞，通过寻找特异性的表面分子标志识别肺癌干细胞，制备单克隆抗体，携带放射性物质或化疗药物，靶向放化疗，最终促使肺癌干细胞凋亡，使肿瘤失去生成新的肿瘤细胞的能力，争取达到根治的目的。这些方法将为肺癌的治疗带来了新的曙光。参考文献 [1]Reya T.Stem cells,cancer,and cancer stem cells.Nature 2001;414(6859):105~111 [2]Wicha MS,Liu S,Dontu G.Cancer stem cells:an old idea a paradigm shift Cancer Res 2006;66(4):1883~1890 [3]Al-Hajj M.Cancer stem cells and oncology therapeutics.Current Opinion in Oncology 2007;19(1):61~64作者单位：154002黑龙江省佳木斯市中心医院
超级一键网克使用方法 进行网克需要的条件： 一键网克软件：超级一键网克，当然其他的网克软件也可以。 一台已经装好系统的机器（操作系统98以上即可，推荐XP）用作服务器， 一台已经装好全部软件并且配置好的机器作为母盘，当然，如果你只对系统盘进行网克的话，这台机器也可以兼做服务器，但是如果你想全盘网克的话，那就需要另外一台机器做服务器了，道理嘛，因为做全盘镜像的时候镜像文件当然不可能放在源盘上，而必须放在另外一块硬盘上，就好象你左手永远不可能摸到左手的手背一样，呵呵。 其它客户机，数量255台以下，当然多于255台也可以，只是下面的IP地址和子网掩码要重新配置，大家参看有关资料。不过我想大家一次网克也不可能会有那么多台机器的，呵呵。 注意事项：首先，这些机器间物理网络要保持畅通（废话！）。另外，如果你的网吧是杂牌军的话，那就不要试了，因为每台机器的配置如果不一样的话，所装的驱动也不一样，否则网克后可能有太多的不可预知的后果，经常蓝屏肯定是家常便饭了，当然也有可能根本就启动不了。以上说的配置主要是显卡和主板（如果你的声卡是集成的话）。硬盘无所谓，但要注意，目标盘的容量一定要大于母盘。当然了，最好是配置完全一样，这样基本上不会出什么问题。
当然，如果你的机器配置不一样的话，也可以进行网克，大家可以把雨林木风ghost版里面的ghost文件提取出来作为镜像，这样也比一台一台装系统要快得多了。不过那应该是分区网克，具体方法你学会了全盘网克那自然不在话下。下面开工。 第一步，制作镜像，如果只制作系统盘的镜像，那么在单机操作就行了。具体操作我想大家都会。下面讲的是制作全盘镜像，当然你不必拆开机器挂两个硬盘了，网克嘛！ 1、配置tftpd32 解压超级一键网克到服务器的任意盘，比如E盘，双击TFTPD32.EXE，关闭防火墙，或者在应用规则里设为允许访问网络。最好关掉，否则下面你要点若干次允许。点DHCP server标签，这里有几个地方要填一下，当前目录和server不要填，程序自动判断。IP地址池随便填，但要注意不要跟服务器的IP地址冲突，当然要符
肺癌干细胞的研究进展 摘要: 肿瘤起源于干细胞，肿瘤干细胞是肿瘤转移、复发的根源。肺癌肿瘤干细胞研究相对滞后，目前为止尚未能获得公认的肺癌肿瘤干细胞数据，但动物实验模型提示肺癌肿瘤干细胞的存在。应用流式细胞仪可分选支气管肺泡干细胞，针对肺癌干细胞的治疗可能是肺癌治疗的新策略。 关键词:肺癌；肿瘤干细胞；支气管肺泡干细胞 Abstract: Tumors come of stem cells,cancer stem are the source of metastasis and recurrent of tumors.The reseaches of lung cancer stem cells relatively fall behind and have not got received data about lung cancer stem cells,but animal experiment models indicate the existence of lung cancer stem https://www.doczj.com/doc/bb12538586.html,e flow cytometer can separate bronchioalveolar stem cells,the treatment to aim directly at lung cancer stem cells may be the new strategy of lung cancer heal. Key words: lung cancer: cancer stem: bronchioalveolar stem cell
目录 摘要 (1) 关键词 (1) Abstract (1) Key word (1) 前言 (1) 1肿瘤干细胞 (1) 1.1干细胞和肿瘤干细胞 (1) 1.2肿干细胞 (2) 2肺癌干细胞 (2) 2.1肺癌干细胞的研究现状 (2) 2.2肺癌干细胞的分选方法 (2) 2.3 肺癌干细胞的鉴别方法 (3) 3肺癌干细胞的研究意义及展望 (3) 参考文献 (4)
实时频谱分析仪（RTSA），这是基于快速傅利叶（FFT）的仪表，可以实时捕获各种瞬态信号，同时在时域、频域及调制域对信号进行全面分析，满足现代测试的需求。 一、实时频谱分析仪的工作原理 在存在被测信号的有限时间内提取信号的全部频谱信息进行分析并显示其结果的仪器主要用于分析持续时间很短的非重复性平稳随机过程和暂态过程，也能分析40兆赫以下的低频和极低频连续信号，能显示幅度和相位。 傅里叶分析仪是实时式频谱分析仪，其基本工作原理是把被分析的模拟信号经模数变换电路变换成数字信号后，加到数字滤波器进行傅里叶分析；由中央处理器控制的正交型数字本地振荡器产生按正弦律变化和按余弦律变化的数字本振信号，也加到数字滤波器与被测信号作傅里叶分析。正交型数字式本振是扫频振荡器，当其频率与被测信号中的频率相同时就有输出，经积分处理后得出分析结果供示波管显示频谱图形。正交型本振用正弦和余弦信号得到的分析结果是复数，可以换算成幅度和相位。分析结果也可送到打印绘图仪或通过标准接口与计算机相连。 二、实时频谱分析仪中的数字信号处理技术 1. IF 数字转换器 一般会数字化以中间频率(IF)为中心的一个频段。这个频段或跨度是可以进行实时分析的最宽的频率范围。在高IF 上进行数字转换、而不是在DC 或基带上进行数字转换，具有多种信号处理优势(杂散性能、DC抑制、动态范围等)，但如果直接处理，可能要求额外的计算进行滤波和分析。 2. 采样 内奎斯特定理指出，对基带信号，只需以等于感兴趣的最高频率两倍的速率取样 3. 具有数字采集的系统中触发 能够以数字方式表示和处理信号，并配以大的内存容量，可以捕获触发前及触发后发生的事件。数字采集系统采用模数转换器(ADC)，在深内存中填充接收的信号时戳。从概念上说，新样点连续输送到内存中，最老的样点将离开内存。
lung cancer stem cells（肺癌干细胞）应用流式细胞仪可分选支气管肺泡干细胞,针对肺癌干细胞的治疗可能是肺癌治疗的新策略。 1、肿瘤干细胞（CSCs）具有自我更新能力。起源于正常干细胞。 干细胞和肿瘤干细胞的相同点：都有无限增殖的能力，可以产生相同的表型和生物学特征不同的子代细胞，有一些相同的信号转导通路，如：WNT、PTEN、Nouth、Hedgehog和BMI1信号通路。 2、正常干细胞——肿瘤干细胞：Wnt、Shh和Notch信号转导异常。它能够使肿瘤不断地繁殖，而且保留原有肿瘤的特征。 3、肿瘤干细胞具有异质性。他们还高表达药物耐药蛋白（如：可以诱导凋亡的蛋白）。 胚胎干细胞也可以无限增殖。 4、肺干细胞( lung stem cells)是指在特定条件下能分化为功能性肺组织,在维持肺组织更新和肺损伤修复中起着重要作用的细胞。具有分化潜能。具有自我更新和高度增值能力。可分为全能干细胞、多能干细胞、胚胎干细胞。 5、支气管肺泡干细胞( bronchioalveolar stem cells,BASC) 该细胞具备克隆增殖、自我更新的能力,同时具有强大的分化能力。 发现一些基因和细胞因子在BASCs恶性转化为肺癌干细胞的过程中起到重要作用: Bmi1(Polycomb家族成员之一)在大多数的上皮肿瘤中都呈现过表达状态,而在肺腺癌中Bmi1的缺失或不足能够通过限制BASCs的扩张潜能从而抑制肿瘤的最初形成[15];肿瘤抑制基因PTEN在正常肺的形态发生、BASCs内环境稳态的维持中发挥作用并能抑制肺腺癌的形成[16]。 6、靶向治疗如果瞄准肺癌干细胞，针对肺癌干细胞的特异性药物的发现将从根本上减少肿瘤复发，提高肿瘤治愈率。 在肿瘤组织中存在少量肿瘤干细胞(cancer stemcells,CSCs),大多处于细胞周
干细胞研究进展消息 干细胞是人体及其各种组织细胞的最初来源, 具有高度自我复制、高度增殖和多向分化的潜能。干细胞研究正在向现代生命科学和医学的各个领域交叉渗透, 干细胞技术也从一种实验室概念逐渐转变成能够看得见的现实。干细胞研究已成为生命科学中的热点。介于此, 本刊将就干细胞的最新研究进展情况设立专栏, 为广大读者提供了解干细胞研究的平台。 干细胞专题近期国外干细胞研究进展 Geron抗癌药GRN163L选择性瞄准癌症干细胞据美国BussinessWire 1月10日报道称, 杰隆(Ge-ron)发表临床前研究数据显示, 其端粒酶抑制剂药物imetelstat (GRN163L)在小儿科神经肿瘤当中可选择性瞄准癌症干细胞, 这一发现为儿童肿瘤的临床试验提供了支持。该研究发表于2011年1月1日的Clinical Cancer Research杂志上。近年来有关端粒酶抑制的研究日益增多, 成为癌症治疗的一个热点方向, GRN163L是此类药物开发中最前沿的一个候选药物。2002年3月, Geron从Lynx Therapcutics获得了用GRN163和GRN163L两种化合物的核心专利。早期研究显示, GRN163对十四种不同癌症细胞均表现出有意义的端粒酶活性抑制作用, 它可以抑制黑色素瘤等细胞的生长。因脂质修饰物GRN163L更易进入细胞发挥端粒酶抑制作用, 后续临床前及临床试验均为GRN163L。2005年, FDA同意GRN163L在患慢性淋巴细胞白血病患者的临床实验。2007年, Geron公司开始GRN163L单独治疗多发性骨髓瘤的I期临床试验。2008年开始了GRN163L治疗乳腺癌的I期临床试验。同年12月, Geron发布了有关GRN163L治疗再发的和难治的多发性骨髓瘤的暂时性临床试验数据。2009年, Geron发布了Geron163L对抗癌症干细胞的实验活动, 包括非小型细胞肺癌、乳癌、胰脏炎、前列腺癌、小儿科神经肿瘤。公司发表Geron163L治疗乳癌的假定癌症干细胞与胰脏炎症系数据。数据显示, 在以Geron163L治疗后, 人类乳癌细胞MCF7的假定干细 胞数量与自我再生的能力大幅减弱。目前Geron163L正处于临床II期试验中。(来源: 生物谷2011-01-11)Cell Stem Cell: iPS细胞具更高基因畸变频率加州大学圣地亚哥分校医学院及斯克里普斯研究所的干细胞科学家领导的跨国研究团队, 记录了在人类胚胎干细胞(hESC)和诱导功能干细胞(iPSC)系中特殊的基因畸变, 研究结果在1月7日的Cell Stem Cell上发表。该公布的发现强调了需要对多能干细胞进行频繁的基因检测以保证其稳定性和临床安全性。该研究的第一作者, 加州大学圣地亚哥分校再生医学系的路易斯·劳伦特博士认为, 人类多能干细胞(hESC和iPSC)比其他类型细胞有更高的基因畸变频率。最令人吃惊的是, 与其他非多能干细胞样本相比较, 观察到hESC的基因扩增和iPSC的缺失方面出现的频率更高。人类多能干细胞在人体内具有发展成其他类型细胞的能力, 可成为细胞替换治疗的潜在来源。斯克里普斯研究员再生医学中心主任珍妮·罗伦教授谈到, 由于基因畸变常常与癌症相关联, 免受癌症相关的基因突变对于临床使用的细胞系来说至关重要。研究团队确认了在多能干细胞系中可能发生突变的基因区域。对于hESC而言, 可观察到的畸变大多是靠近多潜能相关基因区域的基因扩增; 对于iPSC而言, 扩增主要涉及细胞增殖基因及与肿瘤 抑制基因相关的缺失。传统的显微技术, 如染色体组型分析可能无法检测到这些变化。研究组使用一种高分辨率的分子技术, 称为“单核苷酸多态性(SNP)”, 能观察到人类基因组里一百多万个位点里的基因变化。 劳伦特说, 我们惊喜地发现在较短时间培养中的基因变化, 例如在体细胞重编程为多能干细胞的343过程以及在培养中细胞的分化过程。我们不知道这会有怎样的影响, 如果有的话, 这些基因畸变都会对基础研究或者临床应用的结果产生影响, 对此应当深究。劳伦特总结到, 该研究结果解释了有必要对多能干细胞培养进行经常性的基因监控, SNP分析仍不失为人类胚胎干细胞和多能干细胞日常监控的一部分, 但是这一结果提醒我们应当予以重视。(来源: 中国干细胞网2011-01-12)美用胚胎干细胞制造出血小板美国先进细胞技术公司的实验证明, 使用人类胚胎干细胞研制出的血小板可修复实验鼠的受损组织, 人类未来有望源源不断地
一、光盘安装 1、安装盘安装 这是最常规的方法，也是最基本的方法，特点是安全、稳定，缺点是速度慢，需要另外安装驱动程序和软件。简单介绍一下步骤，开机进入BIOS，设置从光驱启动（CD-ROM），放入安装盘，重启电脑，选择相应的菜单，就会进入windows安装程序，选择你想安装的分区，然后根据需要对其进行重分区或格式化操作，然后回车，开始安装。期间会碰到输入密钥、时区、键盘类型等信息，一一输入即可，如果用的是无人职守安装盘，等着就行了，重启几次后，就安好了，记得还要安装驱动。 2、ghost盘安装 GHOST意为幽灵，是赛门铁克公司推出的硬盘分区备份恢复软件，具有简单易行、速度快的特点，一般在ghost光盘启动后会有一键恢复到C盘的选项，也可以进入WINPE，在PE环境下运行GHOST，进行分区恢复。打开GHOST后，先点一下OK，然后依次选择local→partition →from image,找到合适的映像文件，然后选择需要恢复的分区，一般是C盘，确定后等上最多七八分钟就可以了，重启后会进入一个安装程序界面，这是在给你的电脑安装驱动、常用软件的过程，再次重启，系统就装好了。 二、U优盘安装 这个方法适用于没有光驱的电脑。要求电脑支持USB启动，当然，只要不是太旧的电脑，一般都支持的。其中制作相应的启动盘是关键。安装前记得进入BIOS，设置相应的启动方式。 1、优盘启动盘安装
可以是DOS启动盘，或者是PE启动盘，另外需要有GHOST 映像或者光盘映像。开机设置启动方式，这个根据启动盘的模式，可以是USB-HDD、USB-ZIP、USB-FDD等，DOS环境下，打开GHOST，然后还原C盘分区，在PE环境下，也可以打开GHOST 还原分区，这两种方法和用ghost盘安装方法是一样的，只是实现途径不一样。在PE下还可以用虚拟光驱加载光盘映像，然后运行相应的安装程序，开始系统安装，这和安装盘安装方法类似。 2、量产U盘为CD-ROM 准备好U盘、光盘镜像和量产工具，一步一步制作好启动盘，然后，进入BIOS，设置为USB-CDROM启动，随后方法就和光盘安装方法一样了，不过速度要比光盘安装快，这个和优盘读写速度快有关。 三、硬盘安装 这个安装方法几乎不需要什么别的硬件，速度也快。需要可以安装到硬盘的pe，光盘镜像。先在硬盘上安装PE，然后重启，进入PE环境，运行虚拟光驱，加载光盘镜像。还是那样，根据光盘镜像的类型，是安装盘就运行相应的安装程序，是ghost盘就运行ghost恢复C盘分区。 还有就是如果要安装双系统，那么用硬盘安装就方便多了，不用刻盘，也不用制作启动优盘，只要先进入已有的系统，加载光盘镜像，然后运行相应的安装程序就可以了。这里如果是安装盘镜像，启动菜单就已经修改好了，如果是ghost盘，那么，还要使用相应的软件修改启动菜单。
---------------------------------------------------------------最新资料推荐------------------------------------------------------ 干细胞及其研究进展 1 干细胞及其研究进展姓名： 曹晶晶导师： 邓锦波专业: 神经生物学学号： 104753130913 2 干细胞及其研究进展摘要： 干细胞是一类具有自我更新能力的多向分化潜能细胞，在一定 条件下可以分化为多种功能的组织和器官，具有重要的理论研究意 义和临床应用价值。 近年来的研究成果不仅揭示了许多有关细胞生长发育的基础理 论难题，也在创伤修复、神经再生、抵抗衰老、糖尿病、帕金 森氏症、老年痴呆、白血病、肿瘤等疾病的治疗方面显示了巨大 的应用潜力，是应用生物学进入一个崭新的领域。 关键词： 干细胞；分化；诱导性多能干细胞；糖尿病；肿瘤；伦理 争议；正文： 1. 干细胞在人类生命形成的开始，单个受精卵可以分裂发 育形成不同的组织和器官，并通过进一步分裂分化，形成生命个体。 在成体细胞中，大部分高度分化的细胞则失去了再分化的能力， 而特定组织正常的生理代谢或病理损伤也会引起组织或器官的修复 再生，这种具有在分化能力的细胞，即为干细胞。 1 / 17
在一定的条件下，它可以分化成多种功能的器官组织。 这些细胞呈圆形或椭圆形，体积较小，核质比大，具有较强的端粒酶活性，因此具有较强的增殖能力。 干细胞是一种未充分分化、尚不成熟的细胞，其再生各种组织器官和人体的潜在功能，吸引着越来越多人的眼球。 2. 干细胞的研究历史干细胞的研究被认为起始于二十世纪六十年代，加拿大科学家 James E. Till 和 Ernest A. McCulloch 发现并命名造血干细胞之后。 60 年代，几个近亲种系的小鼠睾丸畸胎瘤的研究表明，其来源于胚胎干细胞，确立了胚胎癌细胞是一种干细胞； 1968 年，Edwards 和 Bavister 在体外获得了第一个人卵子； 1978 年，第一个试管婴儿 Louise Brown 在英国诞生。 1981 年， Evan, Kaufman 和 Martin 从小鼠胚泡内细胞群分离出小鼠 ES 细胞，建立了小鼠干细胞体外培养条件，将干细胞注入上鼠，能诱导形成畸胎瘤。 1984-1988 年， Anderews 等人从人睾丸畸胎瘤细胞系 Tera-2 中产生出多能的、克隆化的胚胎癌细胞，克隆的干细胞在视黄酸的作用下分化形成神经元细胞和其他类型的细胞。 1992年， Reynolds和Richards先后在成年鼠的纹状体和海马中分离出神经干细胞。 1996年，轰动世界的polly羊诞生，引发了干细胞研究的热潮。
肿瘤干细胞标志物ABCG2在肺癌中的表达及意义 发表时间：2013-10-24T10:39:06.983Z 来源：《医药前沿》2013年第28期供稿作者：蔡培培田广玉茅国新 [导读] 收集2007年09月～2010年05月间经纤支镜或经皮肺穿刺活检有组织学诊断的Ⅲb期、Ⅳ期的初治肺癌患者的资料，挑选出临床及随访资料完整的病例50例，其中包括非小细胞肺癌43例和小细胞肺癌7例。 蔡培培1 田广玉1 茅国新2（通讯作者） (1扬州市江都人民医院肿瘤科江苏扬州 225200) (2南通大学附属医院肿瘤科江苏南通 226001) 【摘要】目的观察肿瘤干细胞标志物ABCG2在肺癌患者癌组织中的表达情况，并探讨其表达与化疗疗效和预后的关系。方法收集50例 ⅢB或Ⅳ期初治住院肺癌患者的临床资料，其中包括非小细胞肺癌43例和小细胞肺癌7例。应用免疫组化方法检测50例肺癌患者癌组织标本中ABCG2的表达，并分析其表达水平与化疗疗效及无进展生存期(PFS)和总生存期(OS)的关系。结果免疫组化染色显示：ABCG2在非小细胞肺癌中的阳性表达率为62.8%(27/43) 明显高于小细胞肺癌14.3%(1/7)(P＜0.05)，4种不同组织类型肺癌之间比较差异无显著性 (P=0.060)。ABCG2表达阳性组RR明显高于阴性组(P=0.009)。ABCG表达阳性组PFS、OS均显著长于阴性组(P=0.001,P=0.002)。结论肿瘤干细胞标志物ABCG2表达与NSCLC患者预后密切相关，表达阳性提示预后不良，而且ABCG2表达还与化疗疗效有关，ABCG2在NSCLC的发生、发展过程中可能起着重要作用，它有可能成为治疗NSCLC临床耐药的分子靶标。 【关键词】肿瘤干细胞 ABCG2/BCRP1 肺癌 NSCLC 化疗疗效预后 【中图分类号】R730.23 【文献标识码】A 【文章编号】2095-1752（2013）28-0033-02 NCCN推荐含铂类的联合化疗方案为晚期NSCLC的标准治疗方案，但有效率仅为40%左右，且中位生存期<1年[1-2]。原发或继发性耐药的产生是化疗效果不佳的主要原因[3]。许多证据已表明，肿瘤是一种干细胞疾病，干细胞和肿瘤之间有着十分密切的联系，肺癌干细胞及相关研究在肺肿瘤的病因、发病机制、临床诊治及抗肿瘤药物的研究方面具有重要意义。在干细胞和肿瘤细胞中都存在有极少量的侧群(SP) 细胞，SP细胞具备干细胞的多种特性且易于分离，是一个浓缩的干细胞群体，是利用Hoechst染料和流式细胞术进行造血干/祖细胞分离时发现的一群特殊细胞，其表型是由ABC转运蛋白家族介导的，其中一个主要的介导分子为ATP结合转运蛋白G超家族成员2(ABCG2)/乳腺癌耐药蛋白(BCRP1)[4]，可能是肿瘤产生耐药和复发的原因。本研究采用免疫组织化学方法检测正常肺组织和肺癌组织标本中ABCG2的表达情况，并探讨其表达与肺癌临床病理因素之间的相关性及与化疗疗效和预后的关系，以进一步证实肿瘤干细胞学说，从而为肿瘤的靶向治疗提供一定的参考和依据。 1.材料与方法 1.1 研究资料与评析方法 1.1.1 临床资料 收集2007年09月～2010年05月间经纤支镜或经皮肺穿刺活检有组织学诊断的Ⅲb期、Ⅳ期的初治肺癌患者的资料，挑选出临床及随访资料完整的病例50例，其中包括非小细胞肺癌43例和小细胞肺癌7例。并符合以下条件: (1)年龄18～75岁, PS评分≤2分。（2）具有临床可测量病灶。（3）综合胸部CT、腹部CT或腹部B超、骨骼ECT、头颅MRI或PET/CT等全身检查的结果，根据国际抗癌联盟(UICC)提出的TNM分期(2003年修订版)标准对其进行分期，分期为ⅢB或Ⅳ期的不能手术的初治肺癌病人。(4)经纤维支气管镜或CT引导下经皮NCCN推荐含铂类的联合化疗方案为晚期NSCLC的标准治疗方案，但有效率仅为40%左右，且中位生存期<1年[1-2]。原发或继发性耐药的产生是化疗效果不佳的主要原因[3]。许多证据已表明，肿瘤是一种干细胞疾病，干细胞和肿瘤之间有着十分密切的联系，肺癌干细胞及相关研究在肺肿瘤的病因、发病机制、临床诊治及抗肿瘤药物的研究方面具有重要意义。在干细胞和肿瘤细胞中都存在有极少量的侧群 (SP) 细胞，SP细胞具备干细胞的多种特性且易于分离，是一个浓缩的干细胞群体，是利用Hoechst染料和流式细胞术进行造血干/祖细胞分离时发现的一群特殊细胞，其表型是由ABC转运蛋白家族介导的，其中一个主要的介导分子为ATP结合转运蛋白G超家族成员2(ABCG2)/乳腺癌耐药蛋白(BCRP1)[4]，可能是肿瘤产生耐药和复发的原因。本研究采用免疫组织化学方法检测正常肺组织和肺癌组织标本中ABCG2的表达情况，并探讨其表达与肺癌临床病理因素之间的相关性及与化疗疗效和预后的关系，以进一步证实肿瘤干细胞学说，从而为肿瘤的靶向治疗提供一定的参考和依据。 1.材料与方法 1.1 研究资料与评析方法 1.1.1 临床资料 收集2007年09月～2010年05月间经纤支镜或经皮肺穿刺活检有组织学诊断的Ⅲb期、Ⅳ期的初治肺癌患者的资料，挑选出临床及随访资料完整的病例50例，其中包括非小细胞肺癌43例和小细胞肺癌7例。并符合以下条件: (1)年龄18～75岁, PS评分≤2分。（2）具有临床可测量病灶。（3）综合胸部CT、腹部CT或腹部B超、骨骼ECT、头颅MRI或PET/CT等全身检查的结果，根据国际抗癌联盟(UICC)提出的TNM分期(2003年修订版)标准对其进行分期，分期为ⅢB或Ⅳ期的不能手术的初治肺癌病人。(4)经纤维支气管镜或CT引导下经皮肺穿刺获得足够大小的肿瘤组织块。(5)均经病理学检查确诊的肺癌。（6）接受含铂类为基础联合第三代化疗新药化疗方案经静脉滴注至少2个周期，或化疗至病情进展。 记录符合条件的患者姓名、住院号、性别、年龄、组织学类型、TNM分期、分化程度、治疗经过、无进展生存期（PFS）及总生存时间（OS）等临床资料。 1.1.2 疗效评价 疗效评价在化疗2周期后进行，按WHO疗效评价标准评价疗效。完全缓解(CR):全部可测量病灶完全消失持续至少4周；部分缓解(PR):病灶的最大直径与最大垂直横径乘积总和减少50%以上，持续至少4周；病灶稳定(SD):各病灶的最大直径与最大垂直横径乘积总和减少<50%，或增大<25%，持续至少4周且无新病灶出现；疾病进展(PD):各病灶的最大直径与最大垂直横径乘积总和增大>25%，或出现新的病灶。有效率RR定义为CR、PR病例所占所有病例百分比。CR和PR为有效，SD和PD为无效。无进展生存期(PFS)是指从患者开始化疗至PD或最后一次随访的时间(失访患者)。总生存期(OS)是指从从患者开始化疗至患者死亡或最后一次随访的时间(失访患者)。
软盘、光盘、硬盘克隆一个也不能少 经常玩电脑朋友，肯定常常复制软盘，那么如何才能使复制的软盘与源盘文件完全一样，引导信息、文件分配表（FAT）都完全一样呢？甚至将源盘作成IMG映像保存到硬盘上，在需要时再做成软盘呢？ 如何将光盘做成一个文件，以便于存放到硬盘上，或再刻录出与源光盘一模一样的光盘呢？ 如何将硬盘的启动分区做成一个文件，在操作系统出现问题时，能够整盘恢复回去，使操作系统恢复正常，而省去了重新安装操作系统和各种驱动程序的烦恼呢？ 下面分别进行介绍，不过这需要三款工具的帮忙，它们分别是HD-Copy、WinIso、Ghost， 一、软盘的克隆 使用过DOS的朋友大多都使用过HD-COPY，它是德国人Oliver Fromme编写的一个磁盘对拷程序。功能非常强大，可以用作格式化软盘、软盘扩容、修复坏软盘、保存软盘信息、读取坏软盘、清洗软驱磁头等用处。最大的好处是它能将源盘“克隆”成一个映像（IMG格式)，在需要做成软盘时再恢复就可以了。所以从92年问世至今一直都深受人们的喜爱。 HD-COPY的使用非常简单，在DOS下运行HD-Copy，其主界面如图1，菜单中的英文依次为READ（读取）、WRITE(写入）、VERIFY DESTINATION（校验目标盘）、VERIFY SOURCE（校验源盘）、FORMAT DESTINATION（格式化磁盘）、PUT TO FILE（存入映象文件）、GET FROM FILE（读取映象文件）、SPECIAL MENU（特殊菜单）、ABOUT（关于）、Escapr(exit) （退出），菜单栏的右侧是一些开关。 图1
克隆软盘的操作方法是：先启动HD-COPY再插入源盘，选择READ（或按R键）读取源盘信息到缓冲区（缓冲区是程序在内存中划出的一块用作临时数据交换的区域）。结束后再插入目标盘，选择Write（或直接按W）。如果目标盘中以前存有数据，HD-COPY将会问你是否确定覆盖（Y/N），当然是Y，然后程序将执行对拷动作。如果你不想拷贝目标盘，只想将源盘留一个备份（例如借来的软盘）可以在第一步完成后选择PUT TO FILE（按P键），输入你想要存放的路径和文件名。注意的是文件必须是DOS下的8.3格式，就是说文件名不能超过8个英文字母或4个中文字。将来需要的时候可以把你保存的映象文件找来GET FROM FILE（快捷键G）再执行第二步就可以了。 二、光盘的克隆 随着大容量硬盘的普遍采用，人们已经习惯将光盘拷贝成光盘映像文件使用，普遍采用的便是大名鼎鼎的ISO 9660国际标准格式，因此光盘映像文件也简称ISO文件。因为ISO文件保留了光盘中的全部数据信息（包括光盘启动信息），你可以方便地采用常用光盘刻录软件（如Nero Burning-ROM）通过CD-R/RW烧录成光碟，也可以通过虚拟光驱软件(如Daemon-Tools)直接使用。 WinISO 就是一个CD-ROM 映像文件格式转换工具，并且可以直接编辑光盘映像文件！通过WinISO，你可以在映像文件内部添加/删除/重命名/提取文件。你可以将其他格式的映像文件转换为标准的ISO 格式，同时你也可以从你的CD-ROM 中创建ISO 映像文件。将光盘“克隆”成ISO镜像文件的方法如下： 1.启动WinISO之后将欲克隆的光盘放入光驱中，点击菜单“操作”→“从CDROM制作ISO”项，弹出如图2所示的窗口，在其中的“来源CD-ROM”栏选择光盘所在的盘符，在“输出文件”栏设定好存放光盘镜像文件的路径和文件名，同时要在下部选中“ASPI”来制作ISO文件。 图2
·695· 中国肺癌杂志2011年8月第14卷第8期Chin J Lung Cancer, August 2011, Vol.14, No.8 ·综 述· DOI: 10.3779/j.issn.1009-3419.2011.08.11 肺癌干细胞Wnt 信号通路的研究进展 李小江 贾英杰 张文治 张莹 李宝乐 黄敏娜 包芳芳 吴建国 娄怡 【摘要】 Wnt 信号通路在维持肺癌干细胞的增殖和克隆形成方面发挥着重要作用，可通过影响其关键蛋白质抑制肺癌干细胞增殖，为肺癌的治疗提供新的路径。本文旨在通过总结2005年-2010年肺癌干细胞及Wnt 通路的研究现状，探讨Wnt 通路与肺癌干细胞的关系。 【关键词】 Wnt 信号通路；肺肿瘤；干细胞 【中图分类号】 R734.2 The Research Progress about Wnt Pathway of Lung Cancer Stem Cells Xiaojiang LI 1, Yingjie JIA 1, Wenzhi ZHANG 2, Ying ZHANG 1, Baole LI 3, Minna HUANG 1, Fangfang BAO 3, Jianguo WU 2, Yi LOU 3 1 Department of Oncology, the First Teaching Hospital, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China; 2 Nerve Cells Laboratory, Tianjin Neurosurgery Institute, Tianjin 300060, China;3 Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China Corresponding author: Yingjie JIA, E-mail: jiayingjie1616@https://www.doczj.com/doc/bb12538586.html, 【Abstract 】 Being the most critical signaling molecule in the Wnt pathway, the Wnt/β-catenin signaling pathway plays an important role in the maintenance of the cell proliferation and clone formation of lung cancer stem cells. Since it is closely related to the WNT pathway, the proliferation of lung cancer stem cells can be restrained by blocking the WNT pathway or influencing its key protein. Such method provides a new method for the treatment of lung cancer. By summarizing the state of-the-art research of lung cancer stem cells and the Wnt pathway from 2005 to 2010, their relationship is investigated. 【Key words 】 Wnt pathway; Lung neoplasms; Stem cells This work was supported by a grant from National Natural Science Foundation (to Xiaojiang LI)(No.81001578). 本研究受国家自然科学基金项目（No.81001578）资助 作者单位：300193 天津，天津中医药大学第一附属医院肿瘤科（李小江，贾英杰，张莹，黄敏娜）；300060 天津，天津市神经外科研究所神经细胞实验室（张文治，吴建国）；300193 天津，天津中医药大学（ 李宝乐，包芳芳，娄怡 ）（通讯作者：贾英杰，E-mail: jiay-ingjie1616@https://www.doczj.com/doc/bb12538586.html, ） 肺癌是全球发病率最高的恶性肿瘤，己成为人类因癌症死亡的主要原因[1] 。近年来随着干细胞概念被引入肿瘤学的研究，以及多种肿瘤组织和癌细胞系中肿瘤干细胞得到成功分离和鉴定， “肿瘤干细胞”学说应运而生[2]。2007年肺癌干细胞的研究获得突破性进展，Ho 等[3]首次发现利用Hoechst 染料外排法从多种人肺癌细胞系和人肺癌临床样本中分离出的SP 细胞表现出体外高致瘤率、细胞表面的ABC 转运体蛋白表达上调、人端粒末端转移酶表达增高等与干细胞相似的特性，说明这部分SP 细胞具有肿瘤干细胞特性。Wnt 信号传导通路作为细胞信号转导系统之一，与肺癌的发生发展以及在肺癌干细胞特性维持过程中有着重要作用[4] 。Wnt 信号通路的不恰当激 活，参与了肿瘤的发生及其侵袭转移的过程，该通路可能通过作用于肺癌干细胞而促使肺癌细胞复制更新或转移复发。肺癌干细胞Wnt 通路的研究为肺癌治疗提供了新的突破口。1 Wnt 信号通路 Wnt 信号通路因其启动蛋白质Wnt 而得名。Wnt 最初被发现为果蝇分节极性基因，其功能与胚胎发育和蜕变过程中成体翼的形成有关，Nusse 等于1982年在小鼠乳头瘤病毒整合部位发现并报道了int-1基因，随后发现这一基因与果蝇胚胎发育基因wingless 同源，将两者名称简并后该基因被重新命名为Wnt 。越来越多的证据 [5-7] 表明Wnt 信号途径异常激活参与了多种人类肿瘤的发生，此信号转导途径在维持肿瘤干细胞的特性如肿瘤干细胞的数量、耐药性、克隆形成能力、体内成瘤性等方面也有着重要作用。
进行网克需要的条件： 一键网克软件：超级一键网克，当然其他的网克软件也可以。 一台已经装好系统的机器（操作系统98以上即可，推荐XP）用作服务器， 一台已经装好全部软件并且配置好的机器作为母盘，当然，如果你只对系统盘进行网克的话，这台机器也可以兼做服务器，但是如果你想全盘网克的话，那就需要另外一台机器做服务器了，道理嘛，因为做全盘镜像的时候镜像文件当然不可能放在源盘上，而必须放在另外一块硬盘上，就好象你左手永远不可能摸到左手的手背一样，呵呵。 其它客户机，数量255台以下，当然多于255台也可以，只是下面的IP地址和子网掩码要重新配置，大家参看有关资料。不过我想大家一次网克也不可能会有那么多台机器的，呵呵。 注意事项：首先，这些机器间物理网络要保持畅通（废话！）。另外，如果你的网吧是杂牌军的话，那就不要试了，因为每台机器的配置如果不一样的话，所装的驱动也不一样，否则网克后可能有太多的不可预知的后果，经常蓝屏肯定是家常便饭了，当然也有可能根本就启动不了。以上说的配置主要是显卡和主板（如果你的声卡是集成的话）。硬盘无所谓，但要注意，目标盘的容量一定要大于母盘。当然了，最好是配置完全一样，这样基本上不会出什么问题。 当然，如果你的机器配置不一样的话，也可以进行网克，大家可以把雨林木风ghost版里面的ghost文件提取出来作为镜像，这样也比一台一台装系统要快得多了。不过那应该是分区网克，具体方法你学会了全盘网克那自然不在话下。下面开工。 第一步，制作镜像，如果只制作系统盘的镜像，那么在单机操作就行了。具体操作我想大家都会。下面讲的是制作全盘镜像，当然你不必拆开机器挂两个硬盘了，网克嘛！ 1、配置tftpd32
肿瘤干细胞培养技术 随着干细胞生物学以及肿瘤学研究的不断深入，肿瘤干细胞已成为当前肿瘤研究的热点。肿瘤干细胞的体外培养在肿瘤干细胞研究领域具有不可替代的重要地位，通过分离、纯化及培养肿瘤干细胞可以对其生物学特性如异质性、肿瘤的演化、转移和抗药性等进行研究，为肿瘤的早期诊断与治疗提供了新的思路和策略。 肿瘤干细胞的体外培养多采用无血清培养基(serum free medium, SFM)，根据不同的细胞类型加入适当的细胞因子联合培养以防止其分化。肿瘤细胞系或者是临床肿瘤组织联合采用机械和胶原酶消化肿瘤组织得到单细胞悬液，经过流式细胞仪或者免疫磁珠分选的方法得到肿瘤干细胞使用无血清培养基在37℃，5％CO2饱和湿度的条件下进行体外培养，但值得注意的是临床肿瘤组织的获取应该越新鲜越好，最好是在外科手术后1小时内进行处理否则将影响肿瘤干细胞细胞的活性，不利于体外培养。 无血清培养基有很多种，针对不同的细胞类型有专门的无血清营养液出售。无血清培养基具有以下的优点：各批产品之间成分相对明确、质量相对一致；便于控制培养的生理环境；特殊细胞类型的优化配方有利于提高细胞的稳定性，使不同类型的细胞能在最有利于各自生
长的环境中持续传代培养；依据不同类型的细胞、甚至不同的细胞系(株)都可能有各自的无血清培养基。总的来说可以分为基础营养基及附加成分两大部分[1]。基础培养基一般采用人工合成的培养基主要有DMEM、DMEM/F12、UltraCULTURETM、神经干细胞专用无血清培养基、黑色素瘤专用培养基等其中以DMEM/F12(1:1，Invitrogen)最为常用。附加成分是指在基础培养基中加入各种不同细胞生长所需的营养成分，包括①营养因子：胰岛素、转铁蛋白和亚硒酸钠、牛血清白蛋白、B27、L－谷氨酰胺；②细胞因子：白血病抑制因子、表皮生长因子、成纤维细胞生长因子、神经生长因子、血小板衍化生长因子等。使用无血清培养基培养的细胞经胰酶消化传代后不使用血清终止胰酶的作用，可使用0.1％－0.5%大豆胰酶抑制剂(soybean trypsin inhibitor)。 一、肿瘤干细胞培养中几种常见的细胞因子 (一)白血病抑制因子LIF(leukemin inhibitory factor,LIF) 是白介素6(IL-6)细胞因子家族中的一员，是典型的多功能生长因子，具有多种生物学功能：对细胞生长、增殖与分化有着广泛的作用，抑制分化促进干细胞增殖。胚胎干细胞的体外培养需要添加LIF以抑制其分化，维持其多能性。LIF可抑制成纤维细胞生长因子(Fibroblast Growth Factor ,FGF)、β转移生长因子(β-transforming Growth Factor ,TGFβ)和