外源性EGFP和TH基因修饰骨髓基质细胞源神经干细胞帕金森病猴模型脑内移植后的跟踪观察

目的评价外源性酪氨酸羟化酶(TH)和增强型绿色荧光蛋白(EGFP)基因修饰的骨髓基质细胞源神经干细胞(BMSCs-D-NSCs)在猴帕金森病(PD)模型脑内移植后长期存活情况及其功能评定。方法应用MPTP制成偏侧PD猴模型。将构建的pEGFP-hTH转染培养的自体BMSCs- D-NSCs,而后植入PD猴模型脑内,观察其行为学变化,并进行单光子发射计算机体层摄影术(多巴胺转运蛋白显像)和正电子发射计算机体层摄影术(葡萄精代谢显像)检查。用免疫组化法检测移植细胞的存活情况。结果成功构建pEGFP-hTH和pEGFP-C2转染的BMSCs-D-NSCs中TH和EGFP基因均有效表达;细胞移植后5个月PD猴症状仍有所改善。多巴胺转运蛋白显像见细胞移植组术侧纹状体平均标准摄取值低于对侧及健康猴,而高于PD猴术侧。葡萄糖代谢显像见细胞移植组术侧纹状体平均标准摄取值低于对侧及健康猴,而高于PD猴术侧,而细胞移植组间明显差异。免疫组化发现, 细胞移植术后5个月移植区仍存存大量TH、EGFP阳性细胞。结论 pEGFP-hTH和pEGFP-C2转染的BMSCs-D-NSCs存体外和体内均长期表达外源性TH、EGFP基因,基因工程细胞在PD猴脑内存活并发挥治疗作用。

Observation on intracerebrally transplanted BMSCs-D-NSCs modified by TH and EGFP gene in hemiparkinsonism monkey

Objective To evaluate the function and long-term surviving of intracerebrally transplanted neural stem cells (NSCs-D-BMSCs) derived from bone marrow stromal cells and modified by TH gene on monkeys with hemi-parkinsonism. Methods Hemi-parkinsonism models were induced with infusion of MPTP into unilateral common carotid artery. With the technology of gene re-arrangement, hTH gene in pWAV2-TH plasmid was subcloned into pEGFP-C2 to obtain pEGFP-hTH, which then was transfected into NSCs-BMSCs by NucleofectorTM EGFP- and TH-decorated cells were transplanted into PD monkey models. Behavior changes were observed. SPECT (dopamine transporter imaging) and PET (glucose metabolism imaging) were performed on the monkeys with hemi-parkinsonism. Transplanted cells were also examined by immunohistochemistry to determine their living status. Results pEGFP-hTH was tested to be right by enzyme digestion and sequencing. The cells transfected with pEGFP-hTH and pEGFP-C2 were detected in vitro and effectively expressed TH and EGFP. Symptoms of post-transplanted monkeys improved 5 months after transplantation. Through DAT imaging, the uptake values of the transplanted striatums in monkeys with cell transplants were lower than those of the contralateral ones and healthy monkey, but higher than that of lesioned striatums of PD model. The glucose metabolic rates of the transplanted striatum and thalamus in monkeys with cell transplants were lower, compared to that of the healthy monkey and that of the contralateral sides of themselves, but were higher than that of the lesioned striatum and thalamus of PD model. However, there was no significant difference between cell transplant monkeys. Large numbers of TH and EGFP-positive cells were found in the vicinity of transplanted areas by immunohistochemistry or fluorescence microscope. Conclusion NSCs-D-BMSCs cotransfected with pEGFP-hTH and pEGFP-C2 can expression TH and EGFP gene in vitro and in vivo, and even survive for long-term to perform the treating effects on monkeys with hemi-parkinsonism.