核素报告基因显像监测大鼠脑梗死模型中移植的转基因干细胞的实验研究

发布时间：2018-05-20 14:30

本文选题：生物分布 + (131)~I-FIAU　；参考：《华中科技大学》2011年博士论文

【摘要】：目的通过不同的移植途径(原位移植、脑室移植、颈动脉移植、尾静脉移植)将转基因干细胞移植入大鼠脑梗死模型中,采用生物分布、放射自显影、分子生物学及免疫组织化学的方法,在体外间接监测大鼠脑梗死模型中目的基因的表达水平及放射性计数,分析放射性计数与基因表达水平之间的相关性,获得最佳的细胞移植途径及显像的最佳条件。然后采用单光子发射计算机断层扫描仪和小动物正电子发射断层扫描仪进行活体显像,为活体监测细胞及基因治疗疗效提供实验基础。方法 1.SD大鼠骨髓间充质干细胞的培养及鉴定取4周龄的SD大鼠一只,无菌条件下分离双侧股骨和胫骨,去净软组织后,剪开干骺端,用无血清培养基冲洗骨髓腔,收集冲洗液进行离心,1 OOOrpm/min离心5min,弃上清后用含10%胎牛血清的培养基重悬接种到六孔板内,三天进行一次换液,待生长到80%融合时进行消化传代。取3-4代的细胞进行爬片处理,采用免疫组化的方法对其表面抗原CD34、CD44进行鉴定。 2.大鼠脑梗死模型的建立及评定采用线栓法建立大鼠大脑中动脉栓塞模型,麻醉状态下分离大鼠左侧颈总动脉、颈内动脉和颈外动脉,结扎颈总动脉和颈外动脉,于颈总动脉上剪一个小口,将线栓插入到颈内动脉,进入深度距颈内颈外分叉处18mm,阻塞1h后拔出线栓进行再灌注。通过行为学及TTC染色来判定模型成功与否。 3.细胞准备取4-6代处于指数生长期的细胞,按照每孔5×105的密度接种到六孔板,待生长到80%融合时进行病毒感染,感染复数为150,培养箱内孵育2h后换液,继续培养24h后进行消化,移植备用。 4.细胞移植本研究中,在脑梗死24小时后于立体定位仪上进行细胞移植。每只大鼠均移植2×106个转基因的干细胞,移植细胞重悬体积为15～20μl,生物分布实验中,根据不同的移植途径分组如下：①原位移植组(n=5)；②侧脑室移植组(n=5)；③颈动脉移植组(n=5)；④尾静脉移植组(n=5)；⑤以正常大鼠为对照组(n=4)。参考诸葛启钏主译的第三版《大鼠脑立体定位图谱》,梗死侧原位注射点体外标记为前囟前lmm,旁开3mm,进针5mm；侧脑室移植体表标记为前囟后lmm,旁开1.5mm,进针3.5mm；动脉移植采用脑梗侧颈内动脉内注射,移植完毕后结扎动脉血管近心端。放射自显影和活体显像均只采用原位注射途径进行细胞移植。 5.报告探针的核素标记采用Iodogen固相氧化法进行FAU的131Ⅰ标记,标记产物经C-18小柱甲醇纯化,测定标记率和放化纯,并测定纯化产物在新鲜人血清和PBS中24h的稳定性。采用西门子公司的自动合成模块进行合成标记18F-FHBG。 6.生物分布每只大鼠尾静脉注射1.11MBq纯化后的标记物,24h后处死动物,分别取梗死侧脑组织、对侧脑组织、甲状腺、肺、心肌、肝脏、胃、胰腺、脾、肾、肌肉、骨骼、血液和小肠,称湿重并测量放射性γ计数,经放射性衰减校正后,计算每克组织百分注射剂量率(%ID/g),并计算梗死侧脑组织与血液放射性计数的比值。 7.实时定量PCR 分别提取每组梗死侧和对侧脑组织中TK基因的总RNA,按照Ferments说明书进行逆转录合成cDNA。引物：上游5'-CTCACCCTCATCTTCGACCG-3',下游5'-CCTGCAGATACCGCACCGTA-3'。按照上述引物于实时定量PCR仪上扩增45个循环,总反应体系为10μL。利用2-△△CT方法分析不同组间目的基因表达的相对量(CT)。 8. Western-blot蛋白测定往组织中加入裂解液后进行研磨直至组织完全裂解,移至离心管后12000rpm、4℃离心5min,取上清进行蛋白定量测定。测定完毕后进行SDS-PAGE凝胶电泳,根据蛋白Marker指示终止电泳进行转膜,此时开始染色并加入抗体进行免疫反应,完毕后取出膜进行ECL发光、显影和定影。用计算机图形分析软件自动分析图形的大小和灰度。每组实验均重复3次。 9.放射自显影和荧光照相细胞移植按照上述方法进行,只采用原位注射的途径。分组如下：①正常大鼠注射131I-FIAu；②脑梗死模型大鼠注射131I-FIAU:③脑梗死模型大鼠注射131Ⅰ；④移植转基因干细胞的脑梗死模型注射131I-FIAU.分别于131I-FIAU注射后2h、12h、24h和48h处死动物,取脑后立即行冰冻切片,层厚15μm。然后按照说明书进行放射自显影。 10.SPECT显像按照不同的移植途径将细胞移植入大鼠脑梗死模型中,每只模型移植2×106个细胞,每只大鼠注射0.3mCi131I-FIAU,分别于不同的时间进行SPECT显像,放大2倍,矩阵512×512,采集计数120k。 11.Micro-PET/CT显像将细胞在立体定位仪上定向注射到模型脑组织中,分组如下：①脑梗死模型内移植转基因干细胞并注射18F-FHBG:②脑梗死模型内移植正常细胞并注射18F-FHBG；③正常大鼠脑内移植转基因干细胞并注射18F-FHBG:④脑梗死模型直接注射18F-FDG.动物采用气体麻醉后,置于Micro-PET/CT机上进行显像。 12.资料统计所有数据均采用均数±标准差(x±s)表示,数据统计采用SPSS11.5软件进行分析,以p0.05为差异具有统计学意义。结果 1.BMSCs的培养鉴定经过4代的培养,细胞基本纯化,呈梭形集落样生长,免疫组化鉴定细胞表面抗原CD34表达阴性,CD44表达阳性,证明该方法所获得细胞为骨髓间充质干细胞。 2.大鼠脑梗死模型的评定对动物模型的评定采用5分制,所有实验均选择评分在1-3分之间的动物模型,因为该分值范围内的模型最稳定,死亡率最低,各部分实验的组间差异没有统计学意义,P0.05。TTC染色结果显示,梗死区脑组织未染色呈白色,而正常脑组织区域被染成深红色,说明线栓法造模成功。 3.放射性探针的标记 131I-FAU标记率为60.83±1.48%,放化纯为98.01±0.56%,标记产物在人血清及PBS中24小时的稳定性分别为96.12±0.84%和94.74±0.42%。18F-FHBG的标记率和放化纯分别为34.86±2.41%和99.53±0.27%。 4.生物分布所有移植细胞组内两侧脑组织%ID/g的差异具有统计学意义(t=9.00～15.73,P=0.000～0.003),而对照组差异不明显(t=1.51,P=0.182)。组间脑梗死侧脑组织的%ID/g采用单因素方差分析,所有组与对照组之间的差异显著,有统计学意义(P均0.001),原位移植组与其它各组间的差异具有统计学意义(P=0.000-0.027),脑室移植组、动脉移植组与尾静脉移植组组间差异不明显,无统计学意义(P=0.064～0.662)。 5.实时定量PCR 所有移植细胞组内两侧脑组织TK基因表达相对量之间的差异具有统计学意义(t=26.14-122.44,P均0.001),对于梗死侧脑组织中TK基因的表达量,原位移植组与其它各组间的差异具有统计学意义(P=0.000～0.014),其它三组组间差异不明显,无统计学意义(P=0.112～0.364)。采用Pearson相关分析方法,脑组织中TK基因表达的相对量与每克组织百分注射剂量率呈正相关,r=0.971,P0.001。 6.Western blotting 所有移植细胞组内两侧脑组织内TK/β-actin比值的差异具有统计学意义(t=33.10～117.87,P均0.001)。对于组间脑梗死侧脑组织内的TK/β-actin的比值,原位移植组与其它各组间的差异具有统计学意义(P=0.011-0.016),其它各组组间差异不明显,无统计学意义(P=0.141～0.462)。采用Pearson相关分析方法,脑组织内TK/β-actin的比值与每克组织百分注射剂量率呈正相关,r=0.899,P=0.002。 7.放射自显影放射自显影图像显示,在所有实验组中,梗死侧即注射细胞侧脑组织较周围组织和对侧脑组织有明显的放射性浓聚,差异具有统计学意义,P0.05,而在对照组内两侧脑组织的放射性分布未见明显差异,P=0.131～0.552。脑组织内的灰度值随着时间的延长而逐渐减低,实验组内双侧脑组织的灰度比值在24小时达到峰值(6.63)。 8.活体显像在SPECT图像上,不能够清晰的分辨大鼠脑组织结构。18F-FDG Micro-PET/CT显像可以显示脑梗部位的放射性分布明显减低；在梗死模型大鼠原位注射转基因干细胞后,18F-FHBG显像显示在细胞注射部位有明显的放射性浓聚。结论生物分布和放射自显影实验证明131I-FIAU/TK报告基因系统监测脑梗死模型中移植的转基因骨髓间充质干细胞切实可行,并且报告基因与治疗基因的表达量呈很好的正相关,原位移植是基因治疗脑梗死最佳的细胞移植途径。SPECT舌体显像,由于分辨率较低,不能很好的显示大鼠脑组织结构,而Micro-PET/CT可以清晰地监测到移植细胞的位置,并可以评价基因治疗的疗效,为活体监测移植细胞的存活、转归及疗效提供了实验基础。
[Abstract]:objective
Transgenic stem cells were transplanted into the rat cerebral infarction model by different transplantation methods (in situ transplantation, ventricle transplantation, carotid artery transplantation, and tail vein graft). The expression level of target genes in rat cerebral infarction model was indirectly monitored in vitro by biological distribution, autoradiography, molecular biology and immunohistochemistry. The correlation between radioactivity count and gene expression level is analyzed, and the best conditions for cell transplantation and imaging are obtained. Then, single photon emission computed tomography and small animal positron emission tomography are used for living body imaging to provide real monitoring cell and gene therapy effect. The basis of the test.
Method
Culture and identification of 1.SD rat bone marrow mesenchymal stem cells
One of the 4 week old SD rats was isolated from the femur and tibia under aseptic conditions. After removing the soft tissue, the metaphysis was cut open and the marrow cavity was washed with serum-free medium. The rinse fluid was centrifuged for centrifugation. The 1 OOOrpm/min was centrifuged and 5min was centrifuged. After the supernatant, the culture medium containing 10% fetal bovine serum was inoculated into the six hole plate, and a change of liquid was carried out for three days. The cells from 3-4 generations were treated by climbing tablets. The surface antigen CD34 and CD44 were identified by immunohistochemical staining. The cells were passaged by 80%.
Establishment and evaluation of cerebral infarction model in 2. rats
The left carotid artery, internal carotid artery and external carotid artery were separated from the left carotid artery, carotid artery and external carotid artery in the anesthetic state, and a small neck was cut on the common carotid artery and inserted into the internal carotid artery by inserting the thread plug into the internal carotid artery, 18mm and 1h after blocking. Reperfusion. Behavioral and TTC staining were used to determine whether the model was successful or not.
3. cell preparation
The 4-6 generation cells in the exponential growth period were inoculated to the six hole plate according to the density of 5 x 105 per pore, and the virus infection was carried out when the 80% fusion was grown. The number of infection was 150. The incubator incubated for 2h after incubation, and then cultured for 24h to digest and transplant.
4. cell transplantation
In this study, cell transplantation was performed on a stereotaxic after 24 hours of cerebral infarction. 2 x 106 transgenic stem cells were transplanted in each rat. The resuspension volume of the transplanted cells was 15~20 L. In the biological distribution experiment, the following groups were grouped as follows: (1) in situ transplantation group (n=5); (2) lateral ventricle transplantation group (n=5); (3) carotid artery The transplantation group (n=5); (4) the tail vein transplantation group (n=5); (5) the normal rats as the control group (n=4). Refer to the third edition of the main translations of Zhuge's original version, the stereotaxic map of the rat brain, the in situ injection point of the infarction side was marked in front of the anterior fontanelle, the side opened 3mm, and the needle was 5mm; the side ventricle transplantation body surface was marked with the anterior fontanelle LMM, the side opened 1.5mm, the needle 3.5mm; the artery needle 3.5mm; the artery needle 3.5mm; the artery injection 3.5mm; the artery needle 3.5mm; the artery needle 3.5mm; the artery needle 3.5mm; the artery needle 3.5mm; the artery needle 3.5mm; the artery needle 3.5mm; the artery needle 3.5mm; the artery needle 3.5mm; the artery needle 3.5mm; the artery needle 3.5mm; the artery needle 3.5mm; the artery needle 3.5mm; the artery needle 3.5mm; the artery needle 3.5mm; the artery needle 3.5mm; the artery needle 3.5mm; the artery needle 3.5mm; the artery needle 3.5mm; the artery needle 3.5mm; the artery needle 3.5mm; the artery needle 3.5mm; the artery needle 3.5mm; the artery needle 3.5mm; the artery needle 3.5mm; the artery needle 3.5mm; the artery needle 3.5mm; the artery needle 3.5mm; the artery needle 3.5mm; the artery needle 3.5mm; the artery needle 3.5mm; The transplanted cerebral infarction was injected into the internal carotid artery, and the artery was ligated to the proximal end of the heart after the transplantation. The autoradiography and the living imaging were only used for cell transplantation.
5. the nuclide labeling of the probe
The 131 I labeling of FAU was carried out by Iodogen solid phase oxidation method. The labeling product was purified by C-18 column methanol, the labeling rate and the radiochemical purity were measured. The stability of the purified product in the fresh human serum and PBS was determined. The synthetic marker 18F-FHBG. was made by the automatic synthesis module of SIEMENS company.
6. biological distribution
Each rat's tail vein was injected with 1.11MBq purified marker, and the animal was killed after 24h. The infarcted brain tissue was taken to the lateral brain tissue, the thyroid, the lung, the heart, the liver, the stomach, the pancreas, the spleen, the kidney, the muscles, the skeleton, the blood and the small intestine. The weight of the tissue was calculated and the dose rate of each gram of tissue was calculated after the radiation attenuation correction. (%ID/g), and calculate the ratio of cerebral infarction to blood radioactivity counts.
7. real time quantitative PCR
The total RNA of TK gene in each group of infarct side and contralateral brain tissue was extracted, and the cDNA. primers were synthesized according to the Ferments instructions. The upstream 5'-CTCACCCTCATCTTCGACCG-3', the downstream 5'-CCTGCAGATACCGCACCGTA-3'. amplified 45 cycles on the real-time quantitative PCR instrument according to the primers, and the total reaction system was 10 u L. using 2- Delta Delta CT method. The relative amount of target gene expression (CT) between different groups was analyzed.
Determination of 8. Western-blot protein
After the lysate was added to the tissue, it was lapping until the tissue was completely cracked and moved to the centrifuge tube 12000rpm, then centrifuged for 5min at 4 C, and taken the supernatant for quantitative determination. After the determination, the SDS-PAGE gel electrophoresis was completed and the protein Marker indicated the termination of the electrophoretic membrane. At this time, the antibody was added to the antibody for the immune reaction, and then taken. ECL emission, development and fixation were performed. Computer graphics analysis software was used to automatically analyze the size and grayscale of the graphics. Each experiment was repeated 3 times.
9. autoradiography and fluorography
The cell transplantation was carried out according to the above methods. The group was injected in situ only. (1) the normal rats were injected with 131I-FIAu; (2) the rat model rats with cerebral infarction were injected with 131 I of 131I-FIAU: cerebral infarction model rats; (4) the cerebral infarction model of the transplanted transgenic stem cells was injected with 131I-FIAU., 2h, 12h, 24h and 48h after 131I-FIAU injection, respectively. The animals were killed and frozen immediately after the brain. The thickness was 15 micron M. and then autoradiography was carried out according to the instructions.
10.SPECT imaging
The cells were transplanted into the rat model of cerebral infarction in different ways. 2 x 106 cells were transplanted in each model. Each rat was injected with 0.3mCi131I-FIAU. SPECT imaging was performed at different time, 2 times the magnification, and the matrix 512 x 512, and the count 120K. was collected.
11.Micro-PET/CT imaging
The cells were injected into the model brain tissue by stereotaxic, and the groups were grouped as follows: (1) transplanting transgenic stem cells in cerebral infarction model and injected normal cells in 18F-FHBG: cerebral infarction model and injected 18F-FHBG; (3) normal rats were transplanted in brain and injected into 18F-F directly by injection of 18F-FHBG: 4 cerebral infarction model to direct injection of 18F-F DG. animals were anesthetized with gas and placed on Micro-PET/ CT machine for imaging.
12. data statistics
All data were expressed by mean + standard deviation (x + s). Data were analyzed by SPSS11.5 software, and P0.05 was statistically significant.
Result
Culture and identification of 1.BMSCs
After 4 generations of culture, the cells were basically purified and showed spindle colony like growth. The expression of cell surface antigen CD34 was negative, and the expression of CD44 was positive. It proved that the cells obtained by this method were bone marrow mesenchymal stem cells.
Evaluation of cerebral infarction model in 2. rats
The animal model was evaluated by 5 points. All the experiments selected the animal model with a score of 1-3 points, because the model within the range was the most stable and the mortality was the lowest. There was no statistical difference between the experimental groups. The P0.05.TTC staining results showed that the brain tissue in the infarcted area was not stained white, and the normal brain area was located. It was dyed deep red, indicating the success of the thread making method.
3. radioactivity probe markers
The labeling rate of 131I-FAU was 60.83 + 1.48% and the chemoradiation was 98.01 + 0.56%. The stability of the labeling products for 24 hours in human serum and PBS was 96.12 + 0.84% and 94.74 + 0.42%.18F-FHBG, respectively, and the radiochemical purity were 34.86 + 2.41% and 99.53 + 0.27%., respectively.
4. biological distribution
The difference of%ID/g in both sides of the brain tissue in all the transplanted cells was statistically significant (t=9.00 ~ 15.73, P=0.000 ~ 0.003), but the difference in the control group was not significant (t=1.51, P=0.182). The%ID/g of cerebral infarction in the cerebral infarction between the groups was analyzed by single factor analysis of variance, the difference between all groups and the control group was significant (P 0.001), in situ transplantation The difference between the group and the other groups was statistically significant (P=0.000-0.027). There was no significant difference between the group of ventricle transplantation and the group of the artery transplantation group and the tail vein graft group, and there was no statistical significance (P=0.064 to 0.662).
5. real time quantitative PCR
The difference in the relative amount of TK gene expression in both sides of the brain tissue in all the transplanted cells was statistically significant (t=26.14-122.44, P 0.001). The difference in the expression of TK gene in the cerebral infarction was statistically significant (P= 0 to 0.014), and there was no significant difference between the other three groups, and no statistical difference was found between the other groups. Study significance (P=0.112 ~ 0.364). Using Pearson correlation analysis, the relative amount of TK gene expression in the brain tissue is positively correlated with the dose rate per gram per gram of tissue, r=0.971, P0.001.
6.Western blotting
The difference in the ratio of TK/ beta -actin in the bilateral brain tissues in all the transplanted cells was statistically significant (t=33.10 ~ 117.87, P 0.001). The difference in the ratio of TK/ beta -actin in the cerebral infarction between the group and the other groups was statistically significant (P= 0.011-0.016), and there was no significant difference between the other groups, and there was no difference between the other groups. Study significance (P=0.141 ~ 0.462). Using Pearson correlation analysis, the ratio of TK/ beta -actin in the brain tissue was positively correlated with the dose rate per gram per gram of tissue, r=0.899, P=0.002..
7. autoradiography
Autoradiographic images showed that in all experimental groups, the lateral brain tissue of the injected cell was significantly more concentrated than the surrounding tissue and the contralateral brain tissue in the infarct side. The difference was statistically significant, P0.05, but there was no significant difference in the radioactivity distribution between the two sides of the control group. The gray value in the brain tissue of P=0.131 to 0.552. was along with the contrast. The gray ratio of bilateral brain tissue reached a peak at 24 hours (6.63) in the experimental group.
8. living body imaging
On the SPECT image, the.18F-FDG Micro-PET/CT imaging of the rat brain tissue could not be clearly identified. The radioactivity of the brain stem was obviously reduced. After the injection of transgenic stem cells in the rat model, the 18F-FHBG imaging showed that there was a clear concentration of radioactive concentration at the injection site.
conclusion
The biodistribution and autoradiography show that the 131I-FIAU/TK reporter gene system is feasible to monitor the transplanted mesenchymal stem cells in the cerebral infarction model, and the reporter gene is positively correlated with the expression of the therapeutic gene. In situ transplantation is the best way of gene therapy for brain stem cell transplantation,.SPECT body imaging. Because of low resolution, the brain structure of rat can not be displayed well, and Micro-PET/CT can clearly monitor the location of the transplanted cells, and can evaluate the therapeutic effect of gene therapy. It provides an experimental basis for living, prognosis and effect of living body monitoring of transplanted cells.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R743.3;R-332

【参考文献】