促红细胞生成素基因转染人脐血间充质干细胞治疗新生大鼠缺氧缺血性脑损伤初步研究

Intracerebral transplantation of human erythropoietin gene-modified mesenchymal stem cells derived from human umbilical cord blood alleviates hypoxic ischemic brain injury in neonatal rats

目的探讨转染人促红细胞生成素（EPO）基因的人脐血间充质干细胞（MSCs）脑内移植对新生大鼠缺氧缺血性脑损伤（HIBD）的治疗效果。方法采用密度梯度离心法从足月新生儿脐血中分离培养MSCs,流式细胞术（FCM）检测细胞表面抗原标记CD29、CD44、CD105、CD34、CD106和HLA-DR。将pcDNA3-EPO经脂质体介导转染人脐血MSCs,经G418筛选后获得稳定转染人EPO基因的人脐血MSCs,用RT-PCR、Western blot鉴定外源人EPO基因在人脐血MSCs中的表达。42只7d龄SD大鼠在建立HIBD模型后分为3组：空白对照组（A组,n=12）,未转染EPO基因的MSCs移植组（B组,n=12）,转染EPO基因的MSCs移植组（C组,n=18）,在建立HIBD模型后第3天经大脑左侧皮层进行细胞移植。细胞移植术后第7天将C组大鼠随机挑选6只处死后进行脑组织免疫化学检测,观察移植细胞在脑内的存活和迁移情况;于移植术后第1、7、14、21、28天对3组大鼠进行改良神经功能损害评分（mNSS）。结果从足月新生儿脐血中分离培养出MSCs,流式细胞仪检测结果为强表达CD29、CD44、CD105,不表达CD34、CD106、HLA-DR;RT-PCR、Western blot结果显示转染人EPO基因的人脐血MSCs体外能表达EPO;移植组大鼠脑组织免疫化学检测发现移植细胞在脑内能够存活,并以移植点为中心向周围迁移;mNSS结果为：C组大鼠于移植术后第14天开始mNSS即低于A组（P〈0.05or P〈0.01）,B组则于移植术后第21天开始低于A组（P〈0.05）;C组与B组比较,于移植术后第21、28天,C组低于B组（P〈0.05）。结论转染人EPO基因的人脐血MSCs脑内移植术后对新生大鼠HIBD具有较好的治疗作用。

Objective To investigate the therapeutic effect of intracerebral transplantation of human erythropoietin （EPO） gene-modified mesenchymal stem cells （MSCs） derived from human umbilical cord blood （UCB） on hypoxic-ischemic brain damage （HIBD） in neonatal rats.Methods MSCs were isolated from human UCB by density gradient centrifugation,and identified by flow cytometry （FCM）. pcDNA3-EPO was introduced into the human UCB-derived MSCs by lipofectamine and the expression of exogenous EPO was examined by RT-PCR and Western blot. Forty-two 7-day-old SD rats with HIBD were divided into 3 groups：control group （A group,n = 12）,MSCs transplant group （B group,n = 12）,and EPO gene-modified MSCs transplant group（C group,n = 18）. On the 3rd day after HIBD,MSCs were injected into the left cortex of neonatal rats. Seven days after transplantation,6 rats of C group were sacrificed to investigated the survival and migration of the transplanted cells by immunohistochemtry. On the 1st,7th,14th,21st and 28th days after transplantation,nervous function of 3 groups were evaluated by modified neurological severity score （mNSS）. Results MSCs were isolated from full-term newborn UCB. FACS analysis showed that MSCs were positive for CD29,CD44,and CD105 and negative for CD34,CD106,and HLA-DR. The results of RT-PCR and Western blot analyses indicated that the exogenous EPO could be successfully expressed in human UCB-derived MSCs. Immunocytochemistry analysis of brain tissue sections showed that the transplanted human UCB-derived MSCs could survive and migrate around from the center of transplant site. mNSS showed that the score of C group was significantly smaller than control group on the 14th,21st,and 28th days （P〈 0.05 or P〈 0.01）,and mNSS of B group was significantly smaller than control group on the 21st and 28th days （P〈 0.05）. Compared with B group,the score of C group was significantly smaller on the 21st and 28th day（P〈 0.05）. Conclusion Transplantation of EPO gene-modified MSCs is effective to treat neonatal rat HIBD.