间充质干细胞诱导的神经干细胞对脑出血大鼠的神经保护作用

The Neuroprotective Effect of Mesenchymal Stem Cells Induced Neural Stem Cells on Intracerebral Hemorrhage Rats

目的探讨胎盘间充质干细胞(pMSCs)诱导的神经干细胞(iNSCs)对脑出血(ICH)大鼠的神经保护作用。方法利用诱导培养基将胎盘间充质干细胞诱导生成神经干细胞;采用免疫荧光染色鉴定诱导生成的神经干细胞表达神经干细胞相关标记物的情况。SD大鼠随机分为假手术组(Sham组)、脑出血组(ICH组)、诱导的神经干细胞移植组(iNSCs组),构建大鼠纹状体内囊出血模型,24 h后,将诱导的神经干细胞原位移植入脑出血部位。移植7 d后,采用前肢放置实验、转角实验、改良型神经功能缺损评分等方法评估各组的神经功能;评估各组大脑血肿体积率;采用HE及尼氏染色观察各组大脑组织形态变化;采用免疫荧光双标染色观察各组神经元Cleaved-Caspase-3表达情况;采用Western blot检测各组大脑组织超氧化物歧化酶(superoxide dismutase,SOD)蛋白表达水平。结果胎盘间充质干细胞诱导培养7 d后,有大量直径100μm左右、桑葚样或圆球体形的细胞球出现,细胞球中Nestin、GAP-43、Sox-2等神经干细胞标记物高表达。与ICH组比较,iNSCs组的神经功能障碍改善,且大脑血肿体积率降低(P均<0.05)。HE及尼氏(Nissl)染色结果显示,iNSCs组大脑血肿周围的炎细胞浸润数目较ICH组减少,同时,iNSCs组神经元数量增加(P<0.05)。免疫荧光双标染色结果显示,与ICH组比较,iNSCs组大脑血肿周围Cleaved-Caspase-3阳性神经元数目减少(P<0.05);Western blot结果显示,iNSCs组SOD蛋白水平高于ICH组(P<0.05)。结论胎盘间充质干细胞诱导的神经干细胞移植可能通过提高大脑抗氧化能力,减少脑出血大鼠神经元凋亡,进而促进脑出血大鼠的神经损伤修复。

Objective To investigate the neuroprotective effects of placental mesenchymal stem cells(pMSCs)induced neural stem cells(iNSCs)on intracerebral hemorrhage(ICH)rats.Methods The iNSCs were induced from pMSCs with the induction medium.Immunofluorescence staining was used to identify the expression of neural stem cell related markers in iNSCs.SD rats were randomly divided into sham operation group(Sham group),intracerebral hemorrhage group(ICH group),and induced neural stem cell transplantation group(iNSCs group).The SD rats striatocapsular hemorrhage model were established,and 24 h later,the iNSCs were transplanted into the brain hemorrhage site in situ.After 7 days of transplantation,the neurological function was assessed by forelimb placing test,corner turning test and modified neurological severity score in each group.The hematoma volume rate was measured.The morphological changes of brain tissues were observed by HE and Nissl staining.The expression of Cleaved-Caspase-3 of neurons was observed by immunofluorescence double-label staining.The expression level of superoxide dismutase(SOD)in brain tissues was detected by Western blot.Results After 7 days of induction culture of pMSCs,a large number of neurospheres with 100μm diameter nearly and mulberry-like or orb-shaped appeared,and highly expressed the neural stem cells markers of Nestin,GAP-43 and Sox-2.The neurological function was significantly improved in the iNSCs group compared with that in the ICH group,and the rate of brain hematoma volume in the iNSCs group was significantly reduced compared with that in the ICH group(P all<0.05).The results of HE and Nissl staining showed that the inflammatory cell infiltration around the hematoma was reduced and the number of neurons was significantly increased in the iNSCs group,compared with that in the ICH group(P<0.05).Immunofluorescence double-staining results showed that the number of Cleaved-Caspase-3 positive neurons around the brain haematoma was decreased in the iNSCs group compared with that in the ICH group(P<0.05).Western blot results showed that the expression level of SOD protein was higher in the iNSCs group than that in the ICH group(P<0.05).Conclusion The iNSCs transplantation may reduce neuronal apoptosis in ICH rats by increasing the antioxidant capacity of the brain,thereby promoting neural injury repair in ICH rats.