| PurposeGene modified BMSCs were transplanted into the brains of MCAO models via intraparenchymal route, lateral ventricle, carotid artery and tail vein, respectively. The expression of reporter gene (HSV1-tk and EGFP) and therapeutic gene (BDNF) were evaluated by bio-distribution, autoradiography, molecular biology and immunohisto-chemical methods, then analyzing the relative between the expression of reporter gene and radioactive counts and obtaining the optimal way of injection stem cells and time for acquiring images. Finally, single photo emission computed tomography and positron emission computed tomography imaging were performed to monitoring the transplanted cells. The purpose of present study is to provide experimental evidence for monitoring gene therapy in vivo.Methods1. Culture and identification of BMSCsBilateral femur and tibia were isolated under sterile condition from a 4 week-old SD rat. After soft tissue was cleaned, the metaphysis were cut off and the marrow cavity was washed with serum-free medium. The fluid was collected and centrifuged (1000rpm,4℃) for 5 min. Deposit was resuspended with medium containing 10% fetal calf serum, and then was seeded into the 6-well culture plates. The culture medium was renewal once every three days until growth to 80% confluence, then cells were digestion. Surface antigens CD34 and CD44 were identified using immunohistochemical methods.2. Middle Cerebral Artery Occlusion Model (MCAO)According to the method of Kuge et al., the left common carotid artery (CCA), external carotid artery, and internal carotid artery were exposed under anesthesia. A length of 4-0 monofilament nylon suture which tip were rounded by flame heating was inserted from the left CCA into the internal carotid artery, then advanced approximately 18mm intracranially from the CCA bifurcation. Sixty minutes after MCAO, rats were reanesthetized and reperfusion was performed by withdraw of the suture. All animals underwent behavioral test 24 hours after MCAO.3. Cells preparationThe cells in the exponential growth phase were seeded at a density of 5×105 per well of the 6-well culture plates. After reaching confluence, the cells were infected with Ad5-TK-IRES-BDNF-EGFP at an m.o.i. of 150 pfu/cell. BMSCs were exposed to the viral particles at 37℃for 2 hours, then the medium was removed, and the cells were washed once with phosphate-buffered saline (PBS) and recultured with normal medium for 36h, after which the cells were harvested and used for transplantation.4. Cells transplantationCell transplantation was performed one day after MCAO in this study.2×106 cells in 15ul PBS were injected into each MCAO rat over a 5-min period on the stereotaxic apparatus. About bio-distribution experiment, groups is as follows:①Intraparenchymal route group (n=5);②Lateral ventricle group (n=5);③Carotid artery group (n=5);④Tail vein group (n=5);⑤Normal group (n=4). According to the maps of the book compiled by George et al., the stereotaxic coordinates for cell transplantation in group 1 were 1 mm before bregma,3mm lateral to midline and 6mm beneath the skull surface, the stereotaxic coordinates for group 2 were 1 mm behind bregma,1.5 mm lateral to midline and 3.5 mm beneath the skull surface. In autoradiography experiment and imaging in vivo, cells transplantation is same as group 1. All animals were administrated penicillin (200 thousand U daily) im to prevent infection.5. Radiolabelling of probeFAU (2'-fluoro-2'-deoxy-1-β-D-arabinofuranosyl-uracil) was labeled according to the method described by Lan et al. Labeled product was purified with reverse-phase Sep-Pak C-18 column, then labeled efficiency and radiochemical purity were determined. The stability of 131I-FIAU was determined after incubated for 24h in human serum and PBS. 18F-FHBG was synthesized automatically with special modules.6. Bio-distributionThe animals were killed 24 hours after injected 1.11MBq 131I-FIAU each rat. Bilateral brain, thyroid, lung, heart, liver, stomach, pancreas, spleen, kidney, muscle, bone, blood and small intestine were taken and measured their wet weight andγcounts, respectively. After correction by radioactive decay, the %ID/g and the ratio of %ID/g between infarction brain and blood were calculated.7. Real-time quantitative PCRTotal RNA was extracted from bilateral brain tissue, and cDNA was synthesized according to the instructions of Ferments reverse description kit. Primers:upstream 5'-CTCACCCTCATCTTCGACCG-3', downstream 5'-CCTGCAGATACCGCACCGTA-3'. Amplification of 45 cycles were performed on PCR instrument accordance with the above primers, the total reaction system was 10μL.2-ΔΔCT was used to evaluate the gene expression among different groups.8. Western-blottingThe brain tissue was ground until completely cracking, then centrifuged (12000rpm, 4℃) for 5 min. Supernatant was collected for protein quantitative. SDS-PAGE gel electrophoresis was carried on, and after that transfer film and dyeing were performed. The film was removed and ECL lighting, developing and fixing was continued. The size and gray were analyzed automatically with computer graphics analysis software. Each experiment was repeated for 3 times.9. Autoradiography and photofluorography12 hours after cell transplantation, radiolabelled probe were injected via tail vein. The experimental groups were as follows:group 1-131I-FIAU was injected into normal rats (n=5), group 2-131I-FIAU was injected into MCAO rats (n=5), group 3-131I was injected into MCAO rats (n=5), group 4-131I-FIAU was injected into MCAO rats that have been transplanted gene modified BMSCs (n=5). Four rats randomly selected from each group were killed at 2h,12h,24h and 48h after injection, respectively. The brain tissues were removed and ready for frozen section. Autoradiography and photofluorography were performed.10. SPECT imagingGene modified BMSCs (2×106 cells per rat) were transplanted into MCAO models and 0.3 mCi 131I-FIAU was injected. SPECT imaging was performed at different time. Amplification factor:2 times, Matrix:512X512, Acquisition counts:120K.11. Micro-PET/CT imagingThe experimental groups were as follows:①18F-FHBG was injected into MCAO models that have been transplanted gene modified BMSCs;②18F-FHBG was injected into MCAO models that have been transplanted normal BMSCs;③18F-FHBG was injected into normal rats;④18F-FDG was injected into MCAO models. After animals were anesthetized, Micro-PET/CT imaging was performed.12. Statistical analysisData are presented as mean values±standard error, and analyzed with SPSS 11.5 software, The statistical significance level was set at P<0.05.Results1. Culture and identification of BMSCsBMSCs was almost purification after 4 generations, and morphologically spindle-shaped and cloning grow pattern. The cell surface antigen was negative for CD34 and positive for CD44 proved with immunohistochemical method.2. Middle Cerebral Artery Occlusion Model (MCAO)A 5-points degree was used to evaluate the behavior test of MCAO models. Rats with 1-3 points who had a stable ischemia area and almost without ischemia-induced death were chose for all my experiments. In all experiments, there is no statistical difference among the groups about the points, P<0.05. TTC staining showed that infarcted brain tissue was white, and normal was stained dark red, which indicated that the model was successful.3. Radiolabelling of probeRadiolabelled efficiency and radiochemical purity of 131I-FIAU was 60.83±1.48% and 98.01±0.56%, respectively. Its stability in human serum and PBS for 24 hours was 96.12±0.84% and 94.74±0.42%, respectively. Radiolabelled efficiency and radiochemical purity of 18F-FHBG was 34.86±2.41% and 99.53±0.27%, respectively.4. BiodistributionThe %ID/g of infarcted brain tissue after injected BMSCs was higher than that of the contralateral brain tissue in all experimental groups (t=9.00～15.73, P=0.000-0.003), but not in the control group (t=1.51, P=0.182). The %ID/g of infracted brain tissue in the local injection group was 0.12±0.01, which was significantly higher than that in ventricular group (0.05±0.004), carotid group (0.06±0.002), vein injection group (0.06±0.005) and control group (0.005±0.001) (P=0.000～0.027), but no statistical difference could be seen among the other cell-transplanted groups (P=0.064～0.662).5. Real-time quantitative PCRThe expression of TK gene in infarcted brain tissue was higher than that of contralateral brain tissue in all groups (t=26.14～122.44, P<0.001). There was statistically significant difference between local injection group and other groups about TK expression in infarcted brain (P=0.000～0.014), but not in among the other groups (P=0.112～0.364). The CT of TK gene expression from QR-PCR showed a positive correlation with the %ID/g in the brain tissue (r=0.971, P<0.001).6. Western blottingThe ratio of TK/β-actin in infarcted brain tissue was higher than that of contralateral brain tissue in all groups (t=33.10-117.87, P<0.001). There was statistically significant difference between local injection group and other groups about TK/β-actin in infarcted brain (P=0.011-0.016), but not in among the other groups (P=0.141～0.462). The ratio of TK/β-actin on the western-blotting and the %ID/g also correlated well with each other (r=0.899, P=0.002).7. AutoradiographyIn ARG images, higher radioactivity accumulation was seen on the injection site of experimental group than that of the surrounding tissue (P<0.05) and contralateral brain tissue (P<0.001), but there was no significant difference between the bilateral brain in control group (P=0.131～0.552). The gray values progressively decreased along with time, but the ratio of gray values of bilateral brain tissue reached to the peak (6.63) at 24 hours.8. Imaging in vivoIn the SPECT images, we can not clearly distinguish the brain anatomy. 18F-FDG Micro-PET/CT images can show lower radioactive distribution in the area of infarcted brain. After cells were injected into MCAO models, 18F-FHBG Micro-PET/CT was operated, and images show that there was significantly radioactive accumulation in the cells injection site.ConclusionBiodistribution and autoradiography experiments have proved that 131I-FIAU/TK reporter gene system can be used to monitor the transplanted stem cells in the rat MCAO models, the expression between reporter gene and therapeutic gene was a good positive correlation, and local injection was the optimal way for cell transplantation. Duo to low resolution, SPECT imaging could not present subtle anatomy of brain, while Micro-PETCT could clearly detect the location of transplanted cells, which can provide experimental basis for monitoring the fate of cells and therapeutic efficiency.
|
PDF Full Text Request