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Combined Cerenkov luminescence and nuclear ima

Combined Cerenkov luminescence and nuclear ima
Combined Cerenkov luminescence and nuclear ima

Endocrine Journal 2011, 58 (7), 575-583

Molecular imaging methods have been widely used to investigate biological events in vivo , because they enable researchers to study diseases non-inva-sively in living subjects at the molecular level [1-5]. A variety of molecular imaging modalities have been developed to provide functional and anatomical infor-mation of diseases in living small animals and patients. Such modalities could be included in nuclear imaging such as PET and SPECT, optical imaging (biolumines-cence and fluorescence), magnetic resonance imaging, ultrasound, and computed tomography [1, 2, 6].

Optical imaging is a molecular imaging procedure in which light-producing molecules designed to attach

Combined Cerenkov luminescence and nuclear imaging of radioiodine in the thyroid gland and thyroid cancer cells expressing sodium iodide symporter: Initial feasibility study

Shin Young Jeong 1), Mi-Hye Hwang 1), Jung Eun Kim 1), Sungmin Kang 1), Jeong Chan Park 2), Jeongsoo Yoo 2), Jeoung-Hee Ha 3), Sang-Woo Lee 1), Byeong-Cheol Ahn 1) and Jaetae Lee 1)

1) Department of Nuclear Medicine, Kyungpook National University School of Medicine, Daegu 700-422, Korea 2)

Department of Molecular Medicine, Kyungpook National University School of Medicine, Daegu 700-422, Korea 3)

Department of Pharmacology, Kyungpook National University School of Medicine, Daegu 700-422, Korea

abstract. Radioiodine (RI) such as 131I or 124I, can generate luminescent emission and be detected with an optical imaging (OI) device. To evaluate the possibility of a novel Cerenkov luminescence imaging (CLI) for application in thyroid research, we performed feasibility studies of CLI by RI in the thyroid gland and human anaplastic thyroid carcinoma cells expressing sodium iodide symporter gene (ARO-NIS). For in vitro study, FRTL-5 and ARO-NIS were incubated with RI, and the luminometric and CLI intensity was measured with luminometer and OI device. Luminescence intensity was compared with the radioactivity measured with γ-counter. In vivo CLI of the thyroid gland was performed in mice after intravenous injection of RI with and without thyroid blocking. Mice were implanted with ARO-NIS subcutaneously, and CLI was performed with injection of 124I. Small animal PET or γ-camera imaging was also performed. CLI intensities of thyroid gland and ARO-NIS were quantified, and compared with the radioactivities measured from nuclear images (NI). Luminometric assay and OI confirmed RI uptake in the cells in a dose-dependent manner, and luminescence intensity was well correlated with radioactivity of the cells. CLI clearly demonstrated RI uptake in thyroid gland and xenografted ARO-NIS cells in mice, which was further confirmed by NI. A strong positive correlation was observed between CLI intensity and radioactivity assessed by NI. We successfully demonstrated dual molecular imaging of CLI and NI using RI both in vitro and in vivo . CLI can provide a new OI strategy in preclinical thyroid studies.Key words : Cerenkov radiation, Radioiodine, Optical imaging, Thyroid gland, NIS

to specific cells or molecules are injected into the body and then detected by an optical imaging device. The advantages of this approach are its high sensitiv-ity, low-cost, ease of use, relatively high-throughput, and short acquisition time. Recent advances in optical imaging instruments and molecular probes have made it an excellent tool for small animal research [1, 7]. Despite of these advantages, optical imaging has limi-tations in terms of clinical applications because of poor tissue-penetrating abilities and the limited number of optical imaging probes approved by the Food and Drug Administration. On the other hand, nuclear imaging modalities are advantageous in terms of their high sen-sitivity, good tissue penetration, excellent quantifica -tion and easy translation into clinical application [1, 8]. Nuclear imaging modalities and radioiodine (RI) has been widely used in clinical thyroidology and thyroid research for the past several decades. However, nuclear

Received Feb. 2, 2011; Accepted Apr. 11, 2011 as K11E-051Released online in J-STAGE as advance publication May 7, 2011

Correspondence to: Jaetae Lee, M.D, Ph.D., Department of Nuclear Medicine, Kyungpook National University Hospital, Samduk 2-ga 50, Jung-gu, Daegu, 700-721, Korea. E-mail: jaetae@knu.ac.kr

o riginal

?The Japan Endocrine Society

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NIS and GFP proteins were sorted with flow cytometry (FACSorter BD Biosciences, San Jose, CA, USA). Rat thyroid cells (FRTL-5), and ARO cells stably expressing the NIS gene (ARO-NIS) were prepared for cell studies. The isolation, TSH-dependent growth, and basic characteristics of FRTL-5 cells have been previ-ously described elsewhere [13]. Briefly, FRTL-5 cells were cultured in Coon’s modified Ham’s F-12 medium supplemented with 6 hormones (TSH, 2U/L; insulin, 246mU/L; somastatin, 10mg/L; hydrocortisone, 10nM; transferrin, 5mg/L; glycyl-histidyl-lysine, 2.5 mg/L; Sigma, St. Louis, MO, USA), 5% fetal bovine serum (Hyclone, Logan, UT, USA), and 1% penicillin-strep-tomycin (GIBCO, Carlsbad, CA, USA). Cells were maintained at 37?C in a 5% CO 2 atmosphere with a change of medium every 2 to 3 days, and were pas-saged by every 7 days. ARO-NIS cells were grown in RPMI media (Hyclone, Logan, UT, USA) containing 10% fetal bovine serum and 1% penicillin-streptomy-cin at 37?C in a 5% CO 2 atmosphere.

Assessment of NIS gene expression

For confocal microscopic analysis, cells were seeded at 2 x 104 cells in a Laboratory-Tek German borosil-icate cover glass with 8 chambers (Nunc; Nagel Inc. Roskilde, Denmark) and incubated for 24 hr in growth medium. Cells were subsequently washed with phos-phate buffered saline (PBS) and fixed with 1X B/D cytofix (BD Pharmingen, NJ, USA) at 4?C for 30 min. Cells were washed with 200 μL of 1X BD wash buf -fer and then blocked for 1 hr with PBS containing 5% bovine serum albumin. The cells were subsequently incubated with the primary antibody (mouse anti-hNIS, 1:200 dilution, Molecular Probes, Inc., Eugene, OR, USA) at room temperature (RT) for 1 hr, rinsed 3 times with PBS, incubated with fluorescence-labeled secondary antibody (Alexa 588 goat- anti-mouse, 1:150, Millipore, Bedford, MA, USA) at room temper-ature for 40 min, and then rinsed 3 times with PBS. The slides were mounted with Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA, USA), covered with glass cover slips, and examined by use of a laser confocal scanning system with Leica TCS SP2 (Leica, Wetzlar, Germany).

To evaluate the functional expression of NIS gene in FRTL-5 and ARO-NIS cells, cellular uptake of I-125 was measured in triplicate using a modified ver -sion of the procedure described by Weiss et al. [14]. Briefly, FRTL-5 and ARO-NIS cells were plated at 2 x

imaging modalities also have several drawbacks such as low spatial and temporal resolution, and high cost to purchase and maintain instruments. Therefore, its accessibility to basic researchers is limited.

While nuclear and optical imaging are complemen-tary in many aspects, there would be additive advan-tages if we are able to perform optical imaging using a single imaging probe that also can be used for nuclear imaging. Also, there has been no available optical imaging tool yet for use in basic and preclinical thyroid research, even though these strategies could overcome the weakness of each modality [9]. Optical imaging combined with RI in thyroid research can have the advantages of both modalities and would be a new ave-nue for small animal thyroid imaging research, as well as for imaging patients in clinic.

Cerenkov radiation, a well-known phenomenon, is generated when charged particles travel with a veloc-ity at a speed greater than the phase velocity of light in the given medium [10]. The charged particle induces a local polarization along its path through the medium, and radiation is emitted during the return to equilibrium. Recently, I-131 and I-124, which are commonly used for thyroid imaging, were reported to have sufficient energy to result in Cerenkov radiation that can be visualized with sensitive optical imaging equipment [11, 12].

In order to apply bioluminescence imaging in the thyroidology field, we first tried to demonstrate that RI taken into cultured thyroid cells can produce detectable light photons, and that the visible light from RI in the thyroid gland and NIS-expressing tumor can be imaged in living animals with a standard optical imaging tech-nique. The relationship between the light photon emis-sion detected by optical imaging and the radioactivity measured by nuclear imaging technique was investi-gated both in vitro and in vivo .

Materials and Methods

Establishment of stable cells expressing NIS gene Anaplastic thyroid cancer (ARO) cells were pre-pared for transfection with the recombinant lentivirus co-expressing the sodium-iodide symporter (NIS) and green fluorescent protein (GFP) genes driven by phos -phoglycerate kinase (PGK) promoter (pLenti/PGK-NIS-IRES-GFP). After transfection, the cells were cul-tured for several days and then the GFP fluorescence was analyzed with fluorescent microscopy (Nikon Eclipse Ti-S, Nikon Inc., Japan). Cells expressing both

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Cerenkov luminescence imaging of thyroid In vitro luminometric assay and luminescence imag-ing of radioiodine uptake in FRTL-5 and ARO-NIS cells after incubation with I-124

The day before the iodine uptake, FRTL-5 and ARO-NIS cells were plated in 100-mm or 150-mm plates at 1 x 106 to 8 x 106 cells/dish. Radioactivity of the cells was then determined by incubating the cells with 5 mL of HBSS containing 0.5 % bHBSS and 740 kBq of car-rier-free I-124 and 10 μmol/L sodium iodide at 37 °C for 30 min. After incubation with I-124, the cells were washed twice with ice-cold bHBSS and detached with 2 mL trypsin-EDTA. Detached cells were harvested for the in vitro study in a 24-well plate. A 24-well plate containing FRTL-5 and ARO-NIS cells was scanned for 30 sec using a bench top luminometer. The light intensities were calculated by measuring the RLU sig-nal. Using the luminescence setting with no light inter-ference from the excitation lamp, luminescence imag-ing of Cerenkov radiation was acquired from 24-well plates containing 2 mL of a solution of each cell num-ber at various radioactivity levels. Luminescence sig-nals were expressed as photon/sec within an ROI as described previously. After the luminometric assay and CLI, the radioactivity of the cell lysate was mea-sured using an automatic γ-counter.

Animal models

Animal experiments were performed with the approval of the University A nimal Research Committee. Six-week-old female Balb/c nude mice (JA animal Lab, Korea) were maintained under specific pathogen-free conditions. The nude mice were further divided into two groups for the thyroid imaging study: perchlorate blocking and non-blocking groups.

For tumor xenograft mouse model, ARO-NIS cells suspended in PBS were implanted subcutaneously into the right fore flank (0.3x 106 cells), left hind flank (0.9 x 106 cells), and right hind flank (2.7x 106 cells) of each nude mouse. One day after tumor implantation, optical imaging and small-animal PET imaging were performed.

In vivo nuclear and optical imaging of the thyroid and NIS-expressing tumor

For PET imaging of I-124, the mice were placed in a spread-prone position at 6 hr after injection of I-124 (7.4 MBq/0.2 mL of 0.9% NaCl) into the tail vein, and scanned with a small-animal PET scanner (micro-PET ? R4, CTIMI, Knoxville, TN, USA) for 20 min.

105 cells per well in 24-well plates before uptake test. I-125 uptake was determined by incubating the cells with 500 μL of Hank’s balanced salt solution contain -ing 0.5% bovine serum albumin (bHBSS) and 3.7 kBq carrier-free I-125 and 10 μmol/L sodium iodide (spe -cific activity of 740 MBq/mmol) at 37 °C for 30 min. After incubation, the cells were washed twice with ice-cold bHBSS and lysed with 2% sodium dodecyl sul-fate. The radioactivity of the cell lysates was mea-sured using an automatic γ-counter (Cobra II: Canberra Packard, Packard Bioscience, Dreieich, Germany). To identify the relationship between iodine uptake and cell number, the cells were plated in 24-well plates at 0.125 x 106 to 1 x 106 cells/well, and the iodine uptake was measured as described above. For the blocking study, cells were incubated in I-125 medium with or without 100 mM potassium perchlorate. The radioactivity of the cells was then normalized to the total protein con-tent, which was measured using a bicinchoninic acid (BCA) Protein Assay Kit (Pierce, Rockford, IL). The RI uptake was expressed as pmole/mg of protein. In vitro luminometric assay and luminescence imag-ing of I-131 and I-124

Light signal from Cerenkov radiation of I-124 and I-131 was measured first by a luminometric assay and further imaged with optical imaging device (Xenogen IVIS 100, Caliper, Hopkinton, MA, USA). I-131 and I-124 solutions of various concentrations were pre-pared by serial dilution of a stock solution. A 96-well plate containing 100 μL of each RI solution at differ-ent concentrations (46.25, 92.5, 185, 370, 740 kBq) was scanned for 30 sec using a bench top luminometer (SpectraMax L, MDS Analytical Technologies, USA). The light intensity of RI was calculated by measuring the relative luminescence units (RLU). Luminescence imaging was further performed using the optical imag-ing device for a 96-well plate containing 100 μL of I-124 or I-131 at different radioactivities (46.25, 92.5, 185, 370, 740 kBq) at the luminescence setting with no light interference from the excitation lamp. A region-of-interest (ROI) was selected manually over the sig-nal of the well. The area of the ROI was kept constant, and luminescence signals were expressed as photon per second (photon/sec) within an ROI. The radioactivity of the RI was measured using an automatic γ-counter.

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cells. (R 2 = 0.997 for FRTL-5 and 0.998 for ARO-NIS, Figs. 1B and D)

In vitro luminometric assay and luminescence imag-ing of I-131 & I-124

Cerenkov radiation from I-124 or I-131 was eas-ily detected with the luminometer, even with a small activity (37 kBq). I-124, the positron emitter, revealed strong light signals and I-131, the ?- emitter, also pro-duced sufficient luminescence signals, although not as strong as I-124 (Fig. 2A). Luminometric assay revealed dose-dependent increases in luminometric intensity of I-131 and I-124. The light intensity measured by lumi-nometer (RLU) was well correlated with the radioac-tivity measured with the γ-counter (counts per minute; CPM). The emitted light intensity was directly propor-tional to the radioactivity by linear regression analysis. (R 2

= 0.990 for I-124 and 0.997 for I-131)

Light emission by Cerenkov radiation of I-124 or I-131 was well visualized by optical imaging device (Fig. 2B). The minimal activity of I-124 required to be detected clearly by luminescence imaging was 18.5 kBq/mL. The luminescence signal from I-124 was higher than that from the same dose of I-131. Luminescence signal intensities of ROI were well cor-related with the radioactivity for both I-131 and I-124, respectively. Linear regression analysis revealed a strong positive correlation between the luminescence intensity and the radioactivity (R 2 = 0.989 for I-124 and 0.990 for I-131).

In vitro luminometric assay and luminescence imag-ing of FRTL-5 and ARO-NIS cells after incubation with I-124

Cerenkov radiation from I-124 taken in FRTL-5 cells was detected with the luminometer. Light inten-sity measured by luminometer increased with increas-ing number of FRTL-5 cells. Light intensity (RLU) and the radioactivity (CPM) of FRTL-5 cells were pos-itively correlated in FRTL-5 cells by linear regression analysis (R 2 = 0.993).

Light signals from I-124 taken in ARO-NIS cells were also clearly visualized by optical imaging device, and the luminescence signal intensity was well cor-related with the radioactivity of ARO-NIS cells (Fig. 3). The luminescence signals measured over ROI in the optical images were increased by increases in cell number for ARO-NIS cells. There was a strong posi-tive correlation between the intensity of the light signal

The acquired 3-dimensional emission data were recon-structed to temporally framed sinograms by use of Fourier rebinning and an ordered-subsets expectation maximization reconstruction algorithm without attenu-ation correction. Image reconstruction and quantifica -tion were performed with ASIPro software (Concorde Microsystems Inc., Knoxville, TN, USA).

For γ-camera imaging of I-131, the mice were placed in a spread-supine position at 24 hr after the injection of I-131 (7.4 MBq/0.2 mL of 0.9% NaCl) via the tail vein, and planar static images were obtained for 5 min with a dual-head γ-camera equipped with a pinhole collimator (Infinia; GE Medical Systems, Milwaukee, WI, USA). A ROI was drawn over the anterior neck, including the thyroid gland, using Xeleris software (General Electric Medical Systems, Milwaukee, WI, USA). Thyroid radioactivity was corrected by the background activity. Immediately after the PET or γ-camera imaging, biolu -minescence imaging was performed with the IVIS100, equipped with a cooled charged couple detector cam-era for 3 min. The nude mice were anesthetized and placed in a spread-supine position in the dark chamber of the optical molecular imager. Pseudocolor images indicating photon counts were overlaid on photographs of the mice using the Living Image software v. 2.25 (Caliper, Hopkinton, MA, USA). A ROI was selected manually over the signal intensity. Bioluminescence signals are expressed as photons per second per cubic centimeter per steradian (p/sec/cm 2/sr) within an ROI. The area of the ROI was kept constant for all nuclear and optical images. During nuclear or optical imaging, mice were anesthetized by inhalation of a mixture of isoflurane and oxygen.

results

NIS expression in FRTL-5 and ARO-NIS cells

NIS-specific reactivity was detected in cell membrane of FRTL-5 and ARO-NIS cells by immunofluorescence staining, but not in parental ARO cells. The combined image of membrane staining for NIS and DAPI staining for nucleus is presented in Figs. 1A and C.

The I-125 uptake test, which was performed to evalu-ate functional expression of NIS gene in cells, revealed that I-125 uptake in ARO-NIS cells was about 31-fold higher than in ARO cells and was almost completely inhibited by potassium perchlorate (data not shown). The degree of increase in I-125 uptake was positively correlated with cell number in FRTL-5 and ARO-NIS

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Cerenkov luminescence imaging of thyroid further confirmed by nuclear imaging (γ-camera and small-animal PET) (Figs. 4A & 5A). In mice treated with perchlorate, luminescence imaging revealed a faint light signal in the mouse thyroid and nuclear image also showed similar finding (Figs. 4B & 5B). Luminescence intensity and radioactivity measured from the same ROI drawn over thyroid gland were well correlated by linear regression analysis (R 2 = 0.926 for I-131 and 0.991 for I-124).

and the radioactivity of ARO-NIS cells (R 2 = 0.998).Optical and nuclear imaging of the mouse thyroid using I-131 and I-124

Luminescence imaging successfully detected the light emission from neck of each mouse after intrave-nous injection of I-131 and I-124. The region where light emission was detected was supposed to be thy-roid gland on the optical image, and that was finding Fig. 1 Establishment of stable cells expressing NIS and validation of functional activity of NIS (A) Immunofluorescent staining of NIS

in FRTL-5 cells assessed by confocal microscopy. The cell nuclei were visualized with DAPI staining (left upper, blue). NIS expression was mainly detected on the cell membrane by immunostaining with anti-NIS/PE-conjugated secondary antibody (left lower, red). The combined image (right lower) clearly shows the localization of NIS. (B) In vitro radioiodine uptake of FRTL-5 cells. I-125 uptake was measured after incubation in bHBSS containing 3.7kBq carrier-free I-125 and 10 μM NaI. I-125 uptake of FRTL-5 cells increased with increased cell number. (C) Immunofluorescent staining of NIS and EGFP in ARO-NIS cells assessed by confocal microscopy. EGFP expression (upper right, green) was observed throughout cells while NIS expression was mainly detected on the cell membrane by immunostaining with anti- hNIS followed Alexa 568-conjugated goat anti-mouse IgG Ab (lower left, red). The cell nuclei were visualized with DAPI staining (upper left, blue). The combined image (lower right) shows the localization of NIS which was expected to be on the cell membrane. (D) In vitro radioiodine uptake of ARO-NIS cells. I-125 uptake was measured after incubation in bHBSS containing 3.7 kBq carrier-free I-125 and 10 μM NaI. I-125 uptake of ARO-NIS cells increased with increased cell number.

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In vivo I-124 imaging of implanted tumor cells expressing NIS in mice

Luminescence signals from implanted ARO-NIS cells were clearly visualized in the right fore and both hind flanks by optical imaging (Fig. 6A). Luminescence intensity from tumor cells increased with increasing cell number. Light intensity of the tumor in the left hind flank (0.9x106 cells) was 3 times higher than that of the right fore-flank (0.3x106 cells), and that of the right hind flank (2.7x106 cells) was 3 times higher than that of the left hind flank (0.9x106 cells) by quantita-tive analysis. Small animal PET imaging with I-124 also demonstrated photon uptake in implanted ARO-NIS tumor cells in the right fore and both hind flanks, as well as physiological RI accumulation in the thyroid

gland and stomach (Fig. 6B).

Fig. 2 In vitro luminometric assay of I-131 and I-124 (A) Bar graph of relative luminescence units according to radioactivity of

radioiodine. Light intensity increased with increasing radioactivity of radioiodine (I-131 and I-124). (B) Cerenkov luminescence image of 96-well plates containing I-131 and I-124.

Fig. 3 In vivo Cerenkov lumine-scence imaging of radio-iodine

uptake in cells after incubation with I-124. Cerenkov luminescence image of 24-well plates containing ARO-NIS cells after I-124 inoculation.

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Cerenkov luminescence imaging of thyroid Fig. 4 (A) Luminescence and γ-camera images of normal mouse at 24 hr after injection of I-131 (7.4 MBq). Thyroid uptake of I-131 was

successfully demonstrated by both imaging modalities. (B) Luminescence and γ-camera image of thyroid blocked mouse using perchlorate at 24 hr after injection of I-131 (7.4 MBq). Faint thyroid uptake of I-131 was visualized in both imaging modalities.

Fig. 6 Luminescence (A) and small animal PET (B) images of nude mouse implanted with ARO-NIS tumor cells at 6 hr after injection of

I-124 (7.4 MBq). (A) Luminescence images showed radioiodine uptake in implanted ARO-NIS tumor cells of right fore and both hind flanks (right fore flank: 0.3x106; red arrow, left hind flank: 0.9x106; yellow arrow, right hind flank: 2.7x106; white arrow). (B) Small animal PET images demonstrated that uptake in implanted ARO-NIS tumor cells of right fore and both hind flanks (red, yellow and white arrows). Small animal PET images also showed intense radioiodine accumulation in thyroid gland and stomach.

(A)(B)

Fig. 5 (A) Luminescence and small animal PET images of mouse at 6 hours after injection of I-124 (7.4 MBq). Thyroid uptake of I-124 was

successfully demonstrated by both imaging modalities. (B) Luminescence and small animal PET image of thyroid blocked mouse using perchlorate at 6 hr after injection of I-124 (7.4 MBq). Faint thyroid uptake of I-124 was visualized in both imaging modalities.

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vitro and in vivo thyroid research using both positron and ?--emitting RI. An optical imaging device can image objects within several minutes. The acquisition time of optical imaging is thus much shorter than that of nuclear imaging. Furthermore, an optical imaging device can image up to five small animals simultane -ously with a single instrument, while small animal PET and SPECT can only image one mouse at a time.

For application to thyroid research, CLI using RI has some limitations. I-125 was widely used in vitro thyroid cell study due to proper physical characteristics and cost but I-125 is not suitable for CLI due insufficient energy. I-125 decays by γ decay with a maximum energy of 35 keV with relatively long half life. I-124 and I-131 have a sufficient energy for producing Cerenkov radiation. I-124 produces Cerenkov radiation more than I-131 (Fig. 2). Therefore, I-124 expected higher sensitivity in both in vivo and in vitro studies. However, I-124 is available only in limited institutes.

Furthermore, Cerenkov radiation emits a high inten-sity of photons at a wavelength in the blue light region, and its intensity decreases in proportion to 1/λ2 [12, 17]. Therefore, because of the lower abundance of Cerenkov photons in the high tissue-penetrating near-infrared region, limited tissue penetration is expected. In the present study, thyroid gland of ventral position mouse was well visualized in both CLI and nuclear imaging (Figs. 4 and 5). However, thyroid gland of dorsal position mouse was well visualized in nuclear imaging but not in CLI (Fig. 6). These findings suggest CLI can be mainly applied to superficial organ such as thyroid gland in small animal. For tumor xenograft animal study, subcutaneous tumors have been widely used. While optical imaging visualized the tumor region with little background, nuclear imaging showed RI accumulation in internal organs, which were not clearly observed in the optical images presumably due to the absorption of the light signal by the tissue (Fig. 6). Therefore, CLI can provide a clear image of RI uptake in ARO-NIS tumor xenograft animal model, and the therapeutic response to a certain treatment can easily be assessed by quantitative analysis in an ani-mal model with tumor xenograft. Thus, CLI using RI is considered as a suitable technique for in vivo imag-ing studies of thyroid cancer cells expressing NIS in the xenograft model. Since Cerenkov luminescence tomography is under development to overcome the lim-ited tissue penetration of Cerenkov radiation, it can be utilized for in vivo thyroid study in a large animal in the

Discussion

I-131 and I-124 were previously reported to gen-erate luminescence emissions and could be detected with standard optical imaging instruments [15, 16]; however, the present study is the first to demonstrate the potential of CLI using RIs in the in vitro and in vivo application for thyroid studies. In vitro cell study revealed that the measured light intensity by luminom-eter was well correlated with the degree of radioactiv-ity measured by γ-counter, indicating that the CLI sig -nal can be used for quantitative analysis of in vitro RI uptake. These results suggest that the luminometric assay can replace the nuclear technique for radioactiv-ity count, especially in case nuclear instruments are not available. In vitro cell imaging studies also showed that CLI signal intensity was well correlated with RI uptake in NIS-expressing cells. The success of optical imaging with RI is expected to have some impact on thyroid research, because Cerenkov radiation not only provides actual radioactivity of RI taken into cells but also enables simultaneous in vitro imaging of RI accu-mulation, without nuclear imaging instrument.

Our study also suggests that in vivo CLI with RI (I-131 and I-124) has potential for thyroid research in small animals. RI uptake in thyroid gland and NIS-expressing cancer cells were well visualized using optical and nuclear imaging modalities. CLI was well correlated with nuclear imaging.

The relatively poor quantification ability of optical imaging compared to nuclear imaging modalities has been previously reported [1]. However, our quantita-tive results clearly demonstrated specific thyroid spe -cific RI uptake by CLI, and those were well correlated with radioactivity calculated by nuclear imaging stud-ies. These results suggest CLI of RI is not only an easy and fast tool, but also a quantitative method for thy-roid study in small animals. The present study demon-strated that Cerenkov luminescence intensity increases proportionally with increasing implanted tumor cell number in tumor xenograft animal model. This result suggests that the Cerenkov luminescence intensity reflects the tumor cell number. The potential of CLI using RI is also addressed for the diagnosis and therapy monitoring of NIS-expressing cells in this study .

RI is traditionally studied by PET or γ-cameras, which are expensive, hard to maintain and not widely available instruments to many researchers. Therefore, the commonly used optical device can be used for in

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Cerenkov luminescence imaging of thyroid nescence techniques will become a new molecular imag-ing strategy in preclinical thyroid studies and can be a useful technique for translation to clinical applications. V arious potential applications of the new optical imaging modality could be further explored in thyroidology.

acknowledgments

FRTL-5 cells were provided by Prof. Young-Suk Jo (Chungnam National University, Korea) and the pLenti/PGK-hNIS-IRES-GFP vector was provided by Prof. June-Key Chung (Seoul National University, Korea). This work was supported by the Ministry of Education, Science & Technology (MEST) and the National Research Foundation of Korea (NRF) through the Nuclear R&D Program, 2010 (No. 001-7540) and Nuclear Research & Development Program of the National Research Foundation of Korea (NRF) funded by Ministry of Education, Science & Technology (MEST) (grant code: 2010-0017514). The production of I-124 was partially supported by the QURI project of MEST.

near future, with optical tomography. Recently, Li et al . [18] reported that surface measurements of emitted Cerenkov optical photons could be used to reconstruct the radiotracer activity distribution inside an object by modeling the optical photon propagation with the diffusion equation and then reconstructing the optical emission source distribution iteratively.

The present is a feasibility study of CLI for applica-tion to thyroid research. Therefore, we used relatively high radioactivity of RI for CLI and nuclear imaging and does not apply various acquisition time and radio-activity of RI for imaging optimization. Further stud-ies for imaging optimization are needed.

In conclusion, our study demonstrated the potential of Cerenkov luminescence imaging technique of I-131 and I-124 for application in preclinical research about the thyroid gland and thyroid cancer cells expressing NIS both in vitro and in vivo . Based on these results, CLI might add a new imaging platform to the previous molec-ular imaging modalities and provide an invaluable bridge between nuclear and optical imaging. Cerenkov lumi-

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Li C, Mitchell GS, Cherry SR (2010) Cerenkov lumi-nescence tomography for small-animal imaging. Opt Lett 35: 1109-1111.

脐带干细胞综述

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二、分析天平的基本操作

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用于称量某一固定质量的试剂(如基准物质)或试样。适于称量不易吸潮、在空气中能稳定存在的粉末状或小颗粒样品。 A、去皮将干燥的容器置于秤盘上,待显示平衡后按“去皮”键扣除皮重并显示零点 B、加样打开天平门,用药匙将试样抖入容器内,使之达到所需质量。 固定质量称量法注意:若不慎加入试剂超过指定质量,用牛角匙取出多余试剂,直至试剂质量符合指定要求为止。严格要求时,取出的多余试剂应弃去,不要放回原试剂瓶中。操作时不能将试剂散落于天平盘等容器以外的地方,称好的试剂必须定量地由表面皿等容器直接转入接受容器,此即所谓“定量转移”。 ③递减称量法(减量法) 用于称量一定质量范围的样品或试剂。样品易吸水、易氧化或易与二氧化碳等反应时,可选择此法。。 称量步骤:试样的保存——取出盛试样的称量瓶——称出称量瓶质量——敲样——再称出其质量——样品质量——连续称样——称量工作结束 A、试样保存待称样品放于洁净的干燥容器(称量瓶)中,置于干燥器中保存 B、取出称量瓶左手戴手套取出称量瓶或者用折叠成约1cm的纸取出 C、称出称量瓶质量称出称量瓶质量,记录数据 D、敲样将称量瓶取出,在接收容器的上方倾斜瓶身,用称量瓶盖轻敲瓶口上部使试样慢慢落入容器中,瓶盖始终不要离开接受器上方。当倾出的试样接近所需量时,一边继续用瓶盖轻敲瓶口,一边逐渐将瓶身竖直,使粘附在瓶口上的试样落回称量瓶,然后盖好瓶盖,准确称其质量。两次质量之差,即为试样的质量。按上述方法连续递减,可称量多份试样。

精神分裂症的病因及发病机理

精神分裂症的病因及发病机理 精神分裂症病因:尚未明,近百年来的研究结果也仅发现一些可能的致病因素。(一)生物学因素1.遗传遗传因素是精神分裂症最可能的一种素质因素。国内家系调查资料表明:精神分裂症患者亲属中的患病率比一般居民高6.2倍,血缘关系愈近,患病率也愈高。双生子研究表明:遗传信息几乎相同的单卵双生子的同病率远较遗传信息不完全相同 的双卵双生子为高,综合近年来11项研究资料:单卵双生子同病率(56.7%),是双卵双生子同病率(12.7%)的4.5倍,是一般人口患难与共病率的35-60倍。说明遗传因素在本病发生中具有重要作用,寄养子研究也证明遗传因素是本症发病的主要因素,而环境因素的重要性较小。以往的研究证明疾病并不按类型进行遗传,目前认为多基因遗传方式的可能性最大,也有人认为是常染色体单基因遗传或多源性遗传。Shields发现病情愈轻,病因愈复杂,愈属多源性遗传。高发家系的前瞻性研究与分子遗传的研究相结合,可能阐明一些问题。国内有报道用人类原癌基因Ha-ras-1为探针,对精神病患者基因组进行限止性片段长度多态性的分析,结果提示11号染色体上可能存在着精神分裂症与双相情感性精神病有关的DNA序列。2.性格特征:约40%患者的病前性格具有孤僻、冷淡、敏感、多疑、富于幻想等特征,即内向

型性格。3.其它:精神分裂症发病与年龄有一定关系,多发生于青壮年,约1/2患者于20~30岁发病。发病年龄与临床类型有关,偏执型发病较晚,有资料提示偏执型平均发病年龄为35岁,其它型为23岁。80年代国内12地区调查资料:女性总患病率(7.07%。)与时点患病率(5.91%。)明显高于男性(4.33%。与3.68%。)。Kretschmer在描述性格与精神分裂症关系时指出:61%患者为瘦长型和运动家型,12.8%为肥胖型,11.3%发育不良型。在躯体疾病或分娩之后发生精神分裂症是很常见的现象,可能是心理性生理性应激的非特异性影响。部分患者在脑外伤后或感染性疾病后发病;有报告在精神分裂症患者的脑脊液中发现病毒性物质;月经期内病情加重等躯体因素都可能是诱发因素,但在精神分裂症发病机理中的价值有待进一步证实。(二)心理社会因素1.环境因素①家庭中父母的性格,言行、举止和教育方式(如放纵、溺爱、过严)等都会影响子女的心身健康或导致个性偏离常态。②家庭成员间的关系及其精神交流的紊乱。③生活不安定、居住拥挤、职业不固定、人际关系不良、噪音干扰、环境污染等均对发病有一定作用。农村精神分裂症发病率明显低于城市。2.心理因素一般认为生活事件可发诱发精神分裂症。诸如失学、失恋、学习紧张、家庭纠纷、夫妻不和、意处事故等均对发病有一定影响,但这些事件的性质均无特殊性。因此,心理因素也仅属诱发因

脐带血造血干细胞库管理办法(试行)

脐带血造血干细胞库管理办法(试行) 第一章总则 第一条为合理利用我国脐带血造血干细胞资源,促进脐带血造血干细胞移植高新技术的发展,确保脐带血 造血干细胞应用的安全性和有效性,特制定本管理办法。 第二条脐带血造血干细胞库是指以人体造血干细胞移植为目的,具有采集、处理、保存和提供造血干细胞 的能力,并具有相当研究实力的特殊血站。 任何单位和个人不得以营利为目的进行脐带血采供活动。 第三条本办法所指脐带血为与孕妇和新生儿血容量和血循环无关的,由新生儿脐带扎断后的远端所采集的 胎盘血。 第四条对脐带血造血干细胞库实行全国统一规划,统一布局,统一标准,统一规范和统一管理制度。 第二章设置审批 第五条国务院卫生行政部门根据我国人口分布、卫生资源、临床造血干细胞移植需要等实际情况,制订我 国脐带血造血干细胞库设置的总体布局和发展规划。 第六条脐带血造血干细胞库的设置必须经国务院卫生行政部门批准。 第七条国务院卫生行政部门成立由有关方面专家组成的脐带血造血干细胞库专家委员会(以下简称专家委

员会),负责对脐带血造血干细胞库设置的申请、验收和考评提出论证意见。专家委员会负责制订脐带血 造血干细胞库建设、操作、运行等技术标准。 第八条脐带血造血干细胞库设置的申请者除符合国家规划和布局要求,具备设置一般血站基本条件之外, 还需具备下列条件: (一)具有基本的血液学研究基础和造血干细胞研究能力; (二)具有符合储存不低于1 万份脐带血的高清洁度的空间和冷冻设备的设计规划; (三)具有血细胞生物学、HLA 配型、相关病原体检测、遗传学和冷冻生物学、专供脐带血处理等符合GMP、 GLP 标准的实验室、资料保存室; (四)具有流式细胞仪、程控冷冻仪、PCR 仪和细胞冷冻及相关检测及计算机网络管理等仪器设备; (五)具有独立开展实验血液学、免疫学、造血细胞培养、检测、HLA 配型、病原体检测、冷冻生物学、 管理、质量控制和监测、仪器操作、资料保管和共享等方面的技术、管理和服务人员; (六)具有安全可靠的脐带血来源保证; (七)具备多渠道筹集建设资金运转经费的能力。 第九条设置脐带血造血干细胞库应向所在地省级卫生行政部门提交设置可行性研究报告,内容包括:

电子天平使用说明书.

电子天平使用说明书 使用方法 ◎准备 1、将天平安放在稳定及水平的工作台上,避免振动、气流、阳光直射和剧烈的温度波动; 2、安装称盘; 3、接通电源前请确认当地交流电压是否与天平所附的电源适配器所需电压一致; 4、为获得准确的称量结果,在进行称量前天平应接通电源预热30分钟。 ◎电源 1. 天平随机附配交流电源适配器,输入220+22-33V ~ 50Hz 输出9V 300mA 2. 天平选用电池供电时可打开天平底部的电池盖按极性指示装入电池即可,建议使用9伏碱性电池,可连续工作约12小时。 当天平电池供电时,显示屏左上角电量指示框显示段数表明电池的状态(显示3段:电池充足,显示0段:电池耗尽,当电池电量将耗尽时,最后一个显示段闪烁。 ◎开机 在称盘空载情况下按<开/关>键,天平依次进入自检显示(显示屏所有字段短时点亮、型号显示和零状态显示,当天平显示零状态时即可进行称量; 当遇到相关功能键设置有误无法恢复时,按<开/关>键重新开机即可恢复初始设置状态。

◎校准 为获得准确的称量结果,必须对天平进行校准以适应当地的重力加速度。校准应在天平预热结束后进行,遇到以下情况必须使用外部校准砝码对天平进行校准。 1. 首次使用天平称量之前; 2. 天平改变安放位置后。 校准方法与步骤: 1.准备好校准用的标准砝码并确保称盘空载; 2.按<去皮>键:天平显示零状态; 3.按<校准>键:天平显示闪烁的CAL—XXX,(XXX一般为100、200或其它数字,提醒使用相对应的100g、200g或其它规格的标准砝码 4.将标准砝码放到称盘中心位置,天平显示CAL-XXX,等待几秒钟后,显示标准砝码的量值。此时移去砝码,天平显示零状态,则表示校准结束,可以进行称量。如天平不零状态,应重复进行一次校准工作。 ◎称量 天平经校准后即可进行称量,称量时必须等显示器左下角的“○”标志熄灭后才可读数,称量过程中被称物必须轻拿轻放,并确保不使天平超载,以免损坏天平的传感器。 ◎清零或去皮 清零:当天平空载时,如显示不在零状态,可按<去皮>键,使天平显示零状态。此时才可进行正常称量。

古代汉语词类活用例句列举

古代汉语词类活用例句列举 古代汉语词类活用例句列举《郑伯克段于鄢》1、例:壮公生,惊姜氏。P97 惊:用作使动,使。。。惊。2、例:无生民心。P99 生:用作使动,使。。。产生。3、例:若阙地及泉,隧而相见。P101 隧:名词动用。《公孙无知之乱》4、豕立而啼,P109 立:名词作状语,像人一样丫立。〈安之战〉5、皆主?献子。P117 主:名词动用,以。。。为主。6、君无所辱命。P119 辱:动词使动,使。。。受辱。7、从左右,皆肘之。P123 肘:名词使动,表示用胳膊推撞。8、臣辱戎士。123 辱:动词使动。9、人不难以死免其君。P123 免:用作使动,使。。。免于。10、故中御而从齐候。P123 中:方位名词做状语。〈子产说范宣子轻敝〉11、三周华不注。P122 周:

名词动用。12、郑人病之。P129 病:名词用作意动。13、象有齿而焚其身。P130 焚:动词用作使动。14、宣子说,乃轻弊。P130 轻:形容词用作使动,使。。。轻。〈苏秦连横约纵〉15、今先生俨然不运千里而庭教之。P182 远:形容词用作意动。16、明言章理,兵甲愈起。P183 明、章:用作使动。 1 17、辨言伟服。攻战不息。P183 辩、伟:都用作使动,使。。。雄辩,使。。。华美。18、繁称文辞,天下不冶。P183 文:名词用作使动。19、夫徒处而致利,安坐而广地。P183 广:形容词用作使动,使。。。广。20、言语相结,天下为一。P183 言语:名词作状语。21、今欲并天下,凌万乘,诎敌国。制海内,子元元。臣诸候。非兵不可。P183 诎:用作使动,使。。。屈服;子:名词用作使动,使。。。成为子女;臣:名词用作使动,使。。。成为臣子。22、约纵散横,以抑强秦。

分析天平使用说明书

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C.见灵辄饿,问其病,曰:“不食三日矣。”食之,舍其半 D.仓廪实而知礼节,衣食足而知荣辱 四、指出并具体说明下列文句中的词类活用现象: 1.秦数败赵军,赵军固壁不战。(秦与赵兵相距长平) 2.赵王不听,遂将之。(秦与赵兵相距长平) 3.身所奉饭饮而进食者以十数,所友者以百数。(秦与赵兵相距长平) 4.括军败,数十万之众遂降秦,秦悉阬之。(秦与赵兵相距长平) 5.信数与萧何语,何奇之。(韩信拜将) 6.王必欲长王汉中,无所事信。(韩信拜将) 7.吾亦欲东耳,安能郁郁久居此乎?(韩信拜将) 8.何闻信亡,不及以闻,自追之。(韩信拜将) 9.今大王举而东,三秦可传檄而定也。(韩信拜将) 10.遇有以梦得事白上者,梦得于是改刺连州。(柳子厚墓志铭) 11.自子厚之斥,遵从而家焉,逮其死不去。(柳子厚墓志铭) 12.以如司农治事堂,栖之梁木上。(段太尉逸事状) 13.踔厉风发,率常屈其座人。(柳子厚墓志铭) 14.晞一营大噪,尽甲。(段太尉逸事状) 15.即自取水洗去血,裂裳衣疮,手注善药。(段太尉逸事状) 16.黄罔之地多竹,大者如椽。竹工破之,刳去其节,用代陶瓦。(黄冈竹楼记)17.晋灵公不君。厚敛以彫墙。(晋灵公不君) 18.既而与为公介,倒戟以御公徒而免之。(晋灵公不君) 19.盛服将朝,尚早,坐而假寐。(晋灵公不君) 20.晋侯饮赵盾酒,伏甲将攻之。(晋灵公不君) 五、说明下列文句中的词类活用现象,并将全文译为现代汉语:

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2h,以确保仪器始终处于良好使用状态。 4.天平箱内应放置吸潮剂(如硅胶),当吸潮剂吸水变色,应立即高温烘烤更换,以确保吸湿性能。 5.挥发性、腐蚀性、强酸强碱类物质应盛于带盖称量瓶内称量,防止腐蚀天平。 6.称量重量不得过天平的最大载荷。 7.经常对电子天平进行自校或定期外校,保证其处于最佳状态。 8.天平发生故障,不得擅自修理,应立即报告测试中心质量负责人。 9.天平放妥后不宜经常搬动。必须搬动时,移动天平位置后,应由市计量部门校正计量合格后,方可使用。

初中所学文言文中的五类常见词类活用现象

初中所学文言文中的五类常见词类活用现象

古代汉语中的词类活用现象 五种类型:名词用作动词 动词、形容词、名词的使动用法 形容词、名词的意动用法 名词用作状语 动词用作状语 (一)名词用如动词 古代汉语名词可以用如动词的现象相当普遍。如: 从左右,皆肘.之。(左传成公二年) 晋灵公不君.。(左传宣公二年) 孟尝君怪其疾也,衣冠 ..而见之。(战国策·齐策四) 马童面.值,指王翳曰:“此项王也。”(史记·项羽本纪) 夫子式.而听之。(礼记·檀弓下) 曹子手.剑而从之。(公羊传庄公十三年) 假舟楫者,非能水.也,而绝江河。(荀子·劝学) 左右欲刃.相如。(史记·廉颇蔺相如列传) 秦师遂东.。(左传僖公三十二年) 汉败楚,楚以故不能过荥阳而西.。(史记·项羽本纪) 以上所举的例子可以分为两类:前八个例子是普通名词用如动词,后两个例子是方位名词用如动词。 名词用作动词是由上下文决定的。我们鉴别某一个名词是不是用如动词,须要从整个意思来考虑,同时还要注意它在句中的地位,以及它前后有哪些词类的词和它相结合,跟他构成什么样的句法关系。一般情况有如下四种:

①代词前面的名词用如动词(肘之、面之),因为代词不受名词修饰; ②副词尤其是否定副词后面的名词用如动词(“遂东”、“不君”); ③能愿动词后面的名词也用如动词(“能水”、“欲刃”); ④句中所确定的宾语前面的名词用如动词(“脯鄂侯”“手剑”) (二)动词、形容词、名词的使动用法 一、动词的使动用法。 定义:主语所代表的人物并不施行这个动词所表示的动作,而是使宾语所代表的人或事物施行这个动作。例如:《左传隐公元年》:“庄公寤生,惊姜氏。”这不是说庄公本人吃惊,而是说庄公使姜氏吃惊。 在古代汉语里,不及物动词常常有使动用法。不及物动词本来不带宾语,当它带有宾语时,则一定作为使动用法在使用。如: 焉用亡.郑以陪邻?《左传僖公三十年》 晋人归.楚公子榖臣与连尹襄老之尸于楚,以求知罃。(左传成公三年) 大车无輗,小车无杌,其何以行.之哉?《论语·为政》 小子鸣.鼓而攻之可也。《论语·先进》 求也退,故进.之;由也兼人,故退.之。《论语·先进》 故远人不服,则修文德以来.之。《论语·季氏》 有时候不及物动词的后面虽然不带宾语,但是从上下文的意思看,仍是使动用法。例如《论语·季氏》:“远人不服而不能来也”这个“来”字是使远人来的意思。 古代汉语及物动词用如使动的情况比较少见。及物动词本来带有宾语,在形式上和使动用法没有什么区别,区别只在意义上。使动的宾语不是动作的接受者,而是主语所代表的人物使它具有这种动作。例如《孟子·梁惠王上》“朝秦楚”,不食齐宣王朝见秦楚之君,相反的,是齐宣王是秦楚之君朝见自己。 下面各句中的及物动词是使动用法: 问其病,曰:“不食三日矣。”食.之。《左传·宣公二年》

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