131I-FIAU／TK报告基因系统监测大鼠脑梗死模型中移植细胞的研究

Monitoring stem cell transplantation in rat cerebral ischemic infarction model with 131I-FIAU/TKreporter gene system

目的检测经不同途径移植骨髓间充质干细胞后大鼠脑梗死模型中131I一氟一碘阿糖呋喃基尿嘧啶（FIAU）的生物分布及脑组织中胸苷激酶（TK）基因的表达，为核素报告基因显像无创性监测细胞移植治疗脑梗死提供实验依据。方法制备携带TK一内部核糖体连接位点一脑源性神经生长因子（IRES—BDNF）基因的腺病毒重组体Ad5一TK—IRES—BDNF一增强型绿色荧光蛋白（EGFP）；线栓法制作大鼠脑梗死模型；将转基因的干细胞分别通过脑实质、侧脑室、颈动脉和尾静脉注射人大鼠脑梗死模型中，以正常大鼠为对照组；Iodogen固相氧化法标记氟·阿糖呋喃基尿嘧啶（FAU）后进行生物分布研究，计算％ID／g；采用实时定量PCR和Westernblot定量分析TK基因的表达；数据分析采用独立样本t检验、单因素方差分析及Pearson相关分析。结果原位移植组梗死侧脑组织中的％ID／g为0．124±0．013，明显高于侧脑室移植组（0．052±0．004）、颈动脉移植组（0．061±0．002）、尾静脉移植组（0．059±0．005）和对照组（0．005±0．001），差异有统计学意义（t=2．913～5．652，P均〈0．05），其他移植细胞组组间差异无统计学意义（￡=0．694—1．448，P均〉0．05）。所有移植细胞组内两侧脑组织％ID／g的差异有统计学意义（f=9．004～15．734，P均〈0．05），而对照组两侧间差异无统计学意义（t=1．511，P=0．182）。此外，原位移植组梗死侧脑组织中TK基因的表达高于其他各组，差异有统计学意义（t=7．482～12．371，P均〈0．05）；且脑组织TK基因表达的相对量与％ID／g呈正相关（r=0．971，P〈0．001）；脑组织内TK／[3一actin的比值与％ID／g呈正相关（r=0．899，P：0．002）。结论原位注射是治疗脑梗死的最佳细胞移植途径；选择适当示踪剂，可利用PET或SPECT进行无创性活体监测细胞移植治疗脑梗死的效果。

Objective To study the biodistribution of 131 I-2＇-deoxy-l-[3-D-arabinofuranosyl-5-iodo- uracil （FIAU） in the rat middle cerebral artery occlusion model and the expression of thymidine kinase （TK） gene in brain tissue after gene-modified stem cell transplantation, and thus evaluate the possibility of further noninvasive monitoring of stem cell transplantation therapy in cerebral infarction. Methods Adeno- virus recombinant Ad5-TK-internal ribosome entry site-brain derived heurotrophic factor-enhanced green flo- recent protein（IRES-BDNF-EGFP） carrying TK-IRES-BDNF gene was prepared. Cerebral infarction model was established in rats by intraluminal middle cerebral artery occlusion with nylon monofilament. Gene mod- ified bone marrow mesenchymal stem cells were transplanted via intraparenchymal route, lateral ventricle, carotid artery and tail vein, respectively. The normal rats were used as controls. ,31 I- FAU was prepared to be the tracer for biodistribution study and the % ID/g was calculated based on measurement of the tissue ra- dioactivity counts. The expression of TK gene was evaluated by quantitative real-time PCR （QR-PCR） and Western blot analysis. Data were analyzed with independent-samples t-test, one-way analysis of variance （ANOVA） test, and Pearson linear correlation test. Results The % ID/g of infarcted brain tissue in the intraparenchymal group was 0. 124 ± 0. 013, which was significantly higher than that in lateral ventriclegroup （0.052 ± 0. 004 ）, carotid artery group （0.061 ± 0. 002）, tail vein group （0. 059 ± 0. 005 ） and control group （0. 005 -± 0. 001 ） （ t = 2. 913 - 5. 652, all P 〈 0.05 ）, while there were no statistically signifi- cant differences among the other route transplanted groups （ t = 0. 694 - 1. 448, all P 〉 0.05 ）. The differences of % ID/g between the infarcted and contralateral sides of brain tissue in all transplanted groups were statistically significant （ t = 9. 004 - 15. 734, all P 〈 0.05 ）, while there was no statistically signifi- cant difference of this parameter between both sides of brain tissue in control group （t = 1.511, P = 0. 182）. The expression of TK gene in intraparenchymal group was significantly higher than other groups （t = 7.482 - 12. 371, all P 〈0.05 ）. The expression levels of TK gene on QR-PCR showed a positive correlation with % ID/g of the brain tissue （ r = 0. 971, P 〈0.001 ）. Similarly, the ratio of TK/β-αctin by the Western blot analysis correlated with the % ID/g （ r = 0. 899, P = 0. 002）. Conclusion Intraparenchymal route may be the way of choice for cell transplantation therapy of cerebral infarction. If suitable radionuclide tracer is available, PET or SPECT may be potentially used for noninvasive monitoring of stem cell transplantation in cerebral infarc- tion in vivo.