HIF-2α基因下调后的诱导多能干细胞移植对大鼠脑缺血损伤治疗作用的影响

Effect of induced pluripotent stem cell transplantation on treatment of cerebral ischemic injury in rats after down-regulation of HIF-2α gene

目的探讨低氧诱导因子(HIF)-2α基因下调后的诱导多能干细胞(iPSCs)移植对大鼠脑缺血损伤治疗作用的影响。方法 24只雄性SD大鼠随机分为3组(每组8只):iPSCs治疗组、iPSCs+siHIF-2α组,磷酸盐缓冲液(PBS)对照组。所有大鼠均采用大脑中动脉栓塞法制作脑缺血模型,栓塞90 min后进行再灌注。再灌注后3 d进行i PSCs移植、i PSCs+siHIF-2α移植或PBS注射。在移植之前及之后的第2、4周,行神经行为学评价并采用小动物正电子发射体层仪(micro-PET)扫描测定18F-氟代脱氧葡萄糖(18F-FDG)的摄取值。移植后第4周,处死大鼠行免疫荧光检测神经细胞标志物。结果在干细胞移植后第2、4周,i PSCs治疗组的神经功能评分均明显低于PBS对照组(均为P<0.05),而i PSCs+siHIF-2α组的神经功能评分在这两个时间点均高于i PSCs治疗组(均为P<0.05)。在干细胞移植后的第2、4周,与PBS对照组比较,i PSCs治疗组和i PSC+siHIF-2α组脑缺血大鼠脑部的糖代谢水平(损伤侧/正常侧)明显增加(均为P<0.001)。干细胞移植后第2周,i PSCs治疗组大鼠脑缺血区糖代谢水平明显高于iPSCs+siHIF-2α组(P<0.001)。此后iPSCs+siHIF-2α组大鼠脑缺血区糖代谢水平持续上升,至干细胞移植后第4周,与iPSCs治疗组较为接近,但仍低于iPSCs治疗组(P=0.025)。免疫荧光结果提示移植的干细胞存活并迁移至梗死区周边,并且大部分的移植干细胞均表达了神经细胞标志物。结论i PSCs移植对大鼠脑缺血损伤有明显的治疗作用,HIF-2α基因下调对iPSCs的治疗作用有一定的影响。

Objective To investigate the effect of induced pluripotent stem cells(iPSCs)transplantation after the down-regulation of hypoxia-inducible factor(HIF)-2αgene on the treatment of cerebral ischemia injury in rats.Methods Twenty-four male Sprague-Dawley(SD)rats were randomly and evenly divided into the iPSCs treatment,iPSCs+siHIF-2αand phosphate buffer saline(PBS)control groups.The rat models with cerebral ischemia were established by the middle cerebral artery embolization,and reperfusion was carried out 90 min after embolization.At 3 d after reperfusion,iPSCs transplantation,iPSCs+siHIF-2αtransplantation or PBS injection were performed.Before and at 2 and 4 weeks after transplantation,neurobehavioral evaluation was performed and the uptake of 18F-fluoro-deoxyglucose(18F-FDG)was measured by micro-positron emission tomography(micro-PET).At 4 weeks after transplantation,the rats were sacrificed and nerve cell markers were detected by immunofluorescence.Results At 2 and 4 weeks after stem cell transplantation,the neurological function scores of the iPSCs treatment group were significantly lower than those in the PBS control group(both P<0.05),whereas the neurological function scores in the iPSCs+siHIF-2αgroup were considerably higher than those in the iPSCs treatment group(both P<0.05).At 2 and 4 weeks after stem cell transplantation,the glucose metabolism levels(injured side/normal side)in the brain of cerebral ischemia rats in the iPSCs treatment and iPSCs+siHIF-2αgroups were significantly increased compared with those in the PBS control group(both P<0.001).At the 2nd week after stem cell transplantation,the glucose metabolism level in the iPSCs treatment group was significantly higher than that in the iPSCs+siHIF-2αgroup(P<0.001).Subsequently,the glucose metabolism level in the iPSCs+siHIF-2αgroup continued to rise until the 4th week after stem cell transplantation,which was almost identical to but still lower than that in the iPSCs treatment group(P=0.025).Immunofluorescent staining prompted that the transplanted stem cells survived and migrated to the periphery of the infarction area,and a majority of the transplanted stem cells expressed nerve cell markers.Conclusions iPSCs transplantation can be employed to effectively treat cerebral ischemia injury in rats.Down-regulation of HIF-2αgene exerts certain effect upon the therapeutic effect of iPSCs transplantation.