Ang-1基因修饰的骨髓间质干细胞移植治疗脑梗死的实验研究

Therapeutic efficacy of Ang-1 gene-modified MSCs in cerebral infarction

目的:观察血管生成素-1(Ang-1)和骨髓间质干细胞(MSCs)对脑梗死的联合治疗作用。方法:构建带有大鼠Ang-1基因的慢病毒载体,感染大鼠骨髓间质干细胞(rMSCs)获得可表达具有生物活性Ang-1蛋白的rMSCs(Ang-rMSCs)。制备大鼠大脑中动脉栓塞模型,随机分为对照组、Ang-rMSCs组及空载体-rMSCs(pNL-rMSCs)组。脑栓塞术后24h进行细胞移植。各组大鼠分别于术后24h、移植后1周、1月及3月用mNSS量表评价神经功能状况,移植后1周应用伊文思蓝(EB)检测血脑屏障通透性,应用荧光激发及荧光免疫组化观察移植细胞在脑内的存活、分化情况,并经组织学观察有无成瘤迹象。结果:与对照组相比,移植后1周、1月,pNL-rMSCs及Ang-rMSCs组神经功能改善明显(P<0.01),Ang-rMSCs组明显优于pNL-rMSCs组(P<0.001)。移植后3月3组间的mNSS评分差异无显著(P>0.05)。EB检测示血脑屏障通透性在pNL-rMSCs及Ang-rMSCs组低于对照组(P<0.01),Ang-rMSCs组低于pNL-rMSCs组(P<0.01)。移植后1周、1月及3月,Ang-rMSCs和pNL-rMSCs组在荧光激发下均见移植细胞在梗死灶及其周边区聚集并存活。荧光免疫组化检测示Ang-rMSCs组中移植细胞大量表达Ang-1,两移植组中均有部分细胞可同时表达神经元特异性烯醇化酶(NSE)、神经丝蛋白(NF)或星形胶质细胞标志物神经胶质纤维酸性蛋白(GFAP)。未观察到移植细胞异常增殖等成瘤迹象。结论:Ang-1基因修饰的rMSCs移植后可迁移至脑梗死灶周围,分化表达神经细胞标志物并较长期存活。MSCs移植与其分泌的Ang-1蛋白可协同促进脑梗死后神经功能恢复。

AIM： To observe the therapeutic efficacy of Ang - 1 gene - modified mesenchymal stem cells （MSCs） in cerebral infarction. METHODS ： The constructed lentiviral vector carrying the Ang - 1 gene was used to infect the rat mesenchymal stem cells （rMSCs） to establish the Ang - 1 gene - modified rMSCs （Ang - rMSCs）. Adult male F344 rats were subjected to transient （2 h） middle cerebral artery occlusion （MCAO） with modified Zea Longa method.Phosphate buffered saline （ PBS, 1 mL 0. 1 mol/L, for control group ）, or Ang - rMSCs suspension （ 1 mL, for Ang - rMSCs group）, or rMSCs suspension （ 1 mL, for pNL - rMSCs group）, were infused into tail vein of rat respectively at 24 h after MCAO （ n = 8 in each group）. Functional recovery measurements using the modified neurological severity scores （mNSS） were performed at 24 h post - MCAO and 1 week, 1 month and 3 months post - transplantation, respectively. The quantitative evaluation of blood - brain barrier permeability was performed at 1 week post - transplantation. The distribution, differentiation and malignant sign of grafted rMSCs were observed with immunofluorescence staining and histological method. RESULTS ： Significant neurological function improvement was observed in groups treated with Ang - rMSCs or pNL - rMSCs at 1 week, 1 month post - transplantation compared with that in control group, as evidenced by mNSS （P 〈0. 01 ）. Better neurological function improvement was also found in Ang -rMSCs group than that in pNL- rMSCs group （ P 〈 0. 01 ）. The results of quantitative evaluation of blood - brain barrier permeability showed that the permeability in Ang - rMSCs group was the lowest compared to those in other two groups （ P 〈 0. 01 ）, and in the pNL - rMSCs group was the lower than that in control group （ P 〈 0. 01 ）. The grafted rMSCs survived in Ang - rMSCs and pNL - rMSCs groups, most were localized around the ischemic focus, and a few of them expressed NSE, NF and GFAP. The grafted rMSCs expressed BDNF abundantly in Ang - rMSCs group. These grafted rMSCs survived up to 3 months at least. No malignant sign was observed in these grafted cells. CONCLUSION ： Ang - 1 gene - modified MSCs transplantation has better therapeutic efficacy in cerebral infarction than that of MSC transplantation. The transplantation of cells with gene engineering is an effective therapeutic method for stroke patients.