本文選題：生物分布 + (131)~I-FIAU�。� 參考：《華中科技大學》2011年博士論文

【摘要】：目的 通過不同的移植途徑(原位移植、腦室移植、頸動脈移植、尾靜脈移植)將轉(zhuǎn)基因干細胞移植入大鼠腦梗死模型中,采用生物分布、放射自顯影、分子生物學及免疫組織化學的方法,在體外間接監(jiān)測大鼠腦梗死模型中目的基因的表達水平及放射性計數(shù),分析放射性計數(shù)與基因表達水平之間的相關(guān)性,獲得最佳的細胞移植途徑及顯像的最佳條件。然后采用單光子發(fā)射計算機斷層掃描儀和小動物正電子發(fā)射斷層掃描儀進行活體顯像,為活體監(jiān)測細胞及基因治療療效提供實驗基礎(chǔ)。 方法 1.SD大鼠骨髓間充質(zhì)干細胞的培養(yǎng)及鑒定 取4周齡的SD大鼠一只,無菌條件下分離雙側(cè)股骨和脛骨,去凈軟組織后,剪開干骺端,用無血清培養(yǎng)基沖洗骨髓腔,收集沖洗液進行離心,1 OOOrpm/min離心5min,棄上清后用含10%胎牛血清的培養(yǎng)基重懸接種到六孔板內(nèi),三天進行一次換液,待生長到80%融合時進行消化傳代。取3-4代的細胞進行爬片處理,采用免疫組化的方法對其表面抗原CD34、CD44進行鑒定。 2.大鼠腦梗死模型的建立及評定 采用線栓法建立大鼠大腦中動脈栓塞模型,麻醉狀態(tài)下分離大鼠左側(cè)頸總動脈、頸內(nèi)動脈和頸外動脈,結(jié)扎頸總動脈和頸外動脈,于頸總動脈上剪一個小口,將線栓插入到頸內(nèi)動脈,進入深度距頸內(nèi)頸外分叉處18mm,阻塞1h后拔出線栓進行再灌注。通過行為學及TTC染色來判定模型成功與否。 3.細胞準備 取4-6代處于指數(shù)生長期的細胞,按照每孔5×105的密度接種到六孔板,待生長到80%融合時進行病毒感染,感染復數(shù)為150,培養(yǎng)箱內(nèi)孵育2h后換液,繼續(xù)培養(yǎng)24h后進行消化,移植備用。 4.細胞移植 本研究中,在腦梗死24小時后于立體定位儀上進行細胞移植。每只大鼠均移植2×106個轉(zhuǎn)基因的干細胞,移植細胞重懸體積為15～20μl,生物分布實驗中,根據(jù)不同的移植途徑分組如下：①原位移植組(n=5)；②側(cè)腦室移植組(n=5)；③頸動脈移植組(n=5)；④尾靜脈移植組(n=5)；⑤以正常大鼠為對照組(n=4)。參考諸葛啟釧主譯的第三版《大鼠腦立體定位圖譜》,梗死側(cè)原位注射點體外標記為前囟前l(fā)mm,旁開3mm,進針5mm；側(cè)腦室移植體表標記為前囟后lmm,旁開1.5mm,進針3.5mm；動脈移植采用腦梗側(cè)頸內(nèi)動脈內(nèi)注射,移植完畢后結(jié)扎動脈血管近心端。放射自顯影和活體顯像均只采用原位注射途徑進行細胞移植。 5.報告探針的核素標記 采用Iodogen固相氧化法進行FAU的131Ⅰ標記,標記產(chǎn)物經(jīng)C-18小柱甲醇純化,測定標記率和放化純,并測定純化產(chǎn)物在新鮮人血清和PBS中24h的穩(wěn)定性。采用西門子公司的自動合成模塊進行合成標記18F-FHBG。 6.生物分布 每只大鼠尾靜脈注射1.11MBq純化后的標記物,24h后處死動物,分別取梗死側(cè)腦組織、對側(cè)腦組織、甲狀腺、肺、心肌、肝臟、胃、胰腺、脾、腎、肌肉、骨骼、血液和小腸,稱濕重并測量放射性γ計數(shù),經(jīng)放射性衰減校正后,計算每克組織百分注射劑量率(%ID/g),并計算梗死側(cè)腦組織與血液放射性計數(shù)的比值。 7.實時定量PCR 分別提取每組梗死側(cè)和對側(cè)腦組織中TK基因的總RNA,按照Ferments說明書進行逆轉(zhuǎn)錄合成cDNA。引物：上游5'-CTCACCCTCATCTTCGACCG-3',下游5'-CCTGCAGATACCGCACCGTA-3'。按照上述引物于實時定量PCR儀上擴增45個循環(huán),總反應體系為10μL。利用2-△△CT方法分析不同組間目的基因表達的相對量(CT)。 8. Western-blot蛋白測定 往組織中加入裂解液后進行研磨直至組織完全裂解,移至離心管后12000rpm、4℃離心5min,取上清進行蛋白定量測定。測定完畢后進行SDS-PAGE凝膠電泳,根據(jù)蛋白Marker指示終止電泳進行轉(zhuǎn)膜,此時開始染色并加入抗體進行免疫反應,完畢后取出膜進行ECL發(fā)光、顯影和定影。用計算機圖形分析軟件自動分析圖形的大小和灰度。每組實驗均重復3次。 9.放射自顯影和熒光照相 細胞移植按照上述方法進行,只采用原位注射的途徑。分組如下：①正常大鼠注射131I-FIAu；②腦梗死模型大鼠注射131I-FIAU:③腦梗死模型大鼠注射131Ⅰ；④移植轉(zhuǎn)基因干細胞的腦梗死模型注射131I-FIAU.分別于131I-FIAU注射后2h、12h、24h和48h處死動物,取腦后立即行冰凍切片,層厚15μm。然后按照說明書進行放射自顯影。 10.SPECT顯像 按照不同的移植途徑將細胞移植入大鼠腦梗死模型中,每只模型移植2×106個細胞,每只大鼠注射0.3mCi131I-FIAU,分別于不同的時間進行SPECT顯像,放大2倍,矩陣512×512,采集計數(shù)120k。 11.Micro-PET/CT顯像 將細胞在立體定位儀上定向注射到模型腦組織中,分組如下：①腦梗死模型內(nèi)移植轉(zhuǎn)基因干細胞并注射18F-FHBG:②腦梗死模型內(nèi)移植正常細胞并注射18F-FHBG；③正常大鼠腦內(nèi)移植轉(zhuǎn)基因干細胞并注射18F-FHBG:④腦梗死模型直接注射18F-FDG.動物采用氣體麻醉后,置于Micro-PET/CT機上進行顯像。 12.資料統(tǒng)計 所有數(shù)據(jù)均采用均數(shù)±標準差(x±s)表示,數(shù)據(jù)統(tǒng)計采用SPSS11.5軟件進行分析,以p0.05為差異具有統(tǒng)計學意義。 結(jié)果 1.BMSCs的培養(yǎng)鑒定 經(jīng)過4代的培養(yǎng),細胞基本純化,呈梭形集落樣生長,免疫組化鑒定細胞表面抗原CD34表達陰性,CD44表達陽性,證明該方法所獲得細胞為骨髓間充質(zhì)干細胞。 2.大鼠腦梗死模型的評定 對動物模型的評定采用5分制,所有實驗均選擇評分在1-3分之間的動物模型,因為該分值范圍內(nèi)的模型最穩(wěn)定,死亡率最低,各部分實驗的組間差異沒有統(tǒng)計學意義,P0.05。TTC染色結(jié)果顯示,梗死區(qū)腦組織未染色呈白色,而正常腦組織區(qū)域被染成深紅色,說明線栓法造模成功。 3.放射性探針的標記 131I-FAU標記率為60.83±1.48%,放化純?yōu)?8.01±0.56%,標記產(chǎn)物在人血清及PBS中24小時的穩(wěn)定性分別為96.12±0.84%和94.74±0.42%。18F-FHBG的標記率和放化純分別為34.86±2.41%和99.53±0.27%。 4.生物分布 所有移植細胞組內(nèi)兩側(cè)腦組織%ID/g的差異具有統(tǒng)計學意義(t=9.00～15.73,P=0.000～0.003),而對照組差異不明顯(t=1.51,P=0.182)。組間腦梗死側(cè)腦組織的%ID/g采用單因素方差分析,所有組與對照組之間的差異顯著,有統(tǒng)計學意義(P均0.001),原位移植組與其它各組間的差異具有統(tǒng)計學意義(P=0.000-0.027),腦室移植組、動脈移植組與尾靜脈移植組組間差異不明顯,無統(tǒng)計學意義(P=0.064～0.662)。 5.實時定量PCR 所有移植細胞組內(nèi)兩側(cè)腦組織TK基因表達相對量之間的差異具有統(tǒng)計學意義(t=26.14-122.44,P均0.001),對于梗死側(cè)腦組織中TK基因的表達量,原位移植組與其它各組間的差異具有統(tǒng)計學意義(P=0.000～0.014),其它三組組間差異不明顯,無統(tǒng)計學意義(P=0.112～0.364)。采用Pearson相關(guān)分析方法,腦組織中TK基因表達的相對量與每克組織百分注射劑量率呈正相關(guān),r=0.971,P0.001。 6.Western blotting 所有移植細胞組內(nèi)兩側(cè)腦組織內(nèi)TK/β-actin比值的差異具有統(tǒng)計學意義(t=33.10～117.87,P均0.001)。對于組間腦梗死側(cè)腦組織內(nèi)的TK/β-actin的比值,原位移植組與其它各組間的差異具有統(tǒng)計學意義(P=0.011-0.016),其它各組組間差異不明顯,無統(tǒng)計學意義(P=0.141～0.462)。采用Pearson相關(guān)分析方法,腦組織內(nèi)TK/β-actin的比值與每克組織百分注射劑量率呈正相關(guān),r=0.899,P=0.002。 7.放射自顯影 放射自顯影圖像顯示,在所有實驗組中,梗死側(cè)即注射細胞側(cè)腦組織較周圍組織和對側(cè)腦組織有明顯的放射性濃聚,差異具有統(tǒng)計學意義,P0.05,而在對照組內(nèi)兩側(cè)腦組織的放射性分布未見明顯差異,P=0.131～0.552。腦組織內(nèi)的灰度值隨著時間的延長而逐漸減低,實驗組內(nèi)雙側(cè)腦組織的灰度比值在24小時達到峰值(6.63)。 8.活體顯像 在SPECT圖像上,不能夠清晰的分辨大鼠腦組織結(jié)構(gòu)。18F-FDG Micro-PET/CT顯像可以顯示腦梗部位的放射性分布明顯減低；在梗死模型大鼠原位注射轉(zhuǎn)基因干細胞后,18F-FHBG顯像顯示在細胞注射部位有明顯的放射性濃聚。 結(jié)論 生物分布和放射自顯影實驗證明131I-FIAU/TK報告基因系統(tǒng)監(jiān)測腦梗死模型中移植的轉(zhuǎn)基因骨髓間充質(zhì)干細胞切實可行,并且報告基因與治療基因的表達量呈很好的正相關(guān),原位移植是基因治療腦梗死最佳的細胞移植途徑。SPECT舌體顯像,由于分辨率較低,不能很好的顯示大鼠腦組織結(jié)構(gòu),而Micro-PET/CT可以清晰地監(jiān)測到移植細胞的位置,并可以評價基因治療的療效,為活體監(jiān)測移植細胞的存活、轉(zhuǎn)歸及療效提供了實驗基礎(chǔ)。
[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.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R743.3;R-332

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