静脉移植转染GDNF基因的人脐血CD34~+细胞改善局灶性脑缺血大鼠的神经功能

目的 观察静脉移植转染胶质细胞源性神经营养因子(GDNF)基因的人脐血CD34~+细胞对局灶性脑缺血大鼠神经功能的影响及其可能机制.方法 密度梯度离心结合免疫磁珠法收集人脐血CD34~+ 细胞,以携带GDNF基因的质粒转染CD34~+细胞,以空载体转染者作对照.取SD大鼠,线栓法制备大脑中动脉栓塞模型,将模型随机分为3组:治疗组于成模后24 h经尾静脉注射转染GDNF基因的CD34~+细胞1×10~6个;阳性对照组经尾静脉注射空载体转染的CD34~+细胞1×10~6个;空白对照组经尾静脉注射等量生理盐水.另以正常SD大鼠为假手术组.移植后采用改良的神经功能缺损评分(mNSS)评价神经功能恢复状况;图像分析法检测脑梗死体积;酶联免疫吸附试验检测细胞培养液与脑组织匀浆中GDNF水平;免疫组化方法检测CD34~+细胞及其人神经胶质纤维酸性蛋白(GFAP)和人神经元核抗原(NeuN)表达.结果 转染GDNF基因的CD34~+细胞培养上清液中GDNF的含量明显高于空载体转染者(P<0.05).移植后7d,治疗组、阳性对照组和空白对照组间两两比较,mNSS评分的差异均无统计学意义(P>0.05);至移植后28 d,治疗组运动功能改善,mNSS评分为5.0±1.0,显著低于阳性对照组的5.9±1.4和空白对照组的7.0±1.7(P<0.05,P<0.05).移植后28 d,治疗组梗死脑组织体积为(142±44)mm~3,显著低于阳性对照组的(196±58)mm~3(P<0.05)和空白对照组的(233±50)mm~3(P<0.01).移植后28 d,治疗组NeuN阳性细胞为(6.7±2.0)个,GFAP阳性细胞为(14.1±3.3)个,明显高于阳性对照组和空白对照组(P<0.05,P<0.05),而在空白对照组和假手术组未见上述阳性细胞.移植后第28天,治疗组脑组织匀浆中GDNF水平较阳性对照组明显升高(P<0.05).结论 静脉移植转染GDNF基因的人脐血CD34~+细胞可改善局灶性脑缺血大鼠的神经功能,效果优于单纯CD34~+细胞移植,脑组织中GDNF水平的高低可能是导致疗效差异的机制之一.

Intravenous transplantation of GDNF gene-modified CD34~+ cells from human umbilical cord blood for cerebral ischemie-reperfusion inury in rats

Objective To observe the neurologicaI functional recovery after intravenous transplantation of human umbilical cord blood(HUCB)CD34~+ cells transfected with glial cell-derived neurotrophic factor (GDNF) and green fluorescent protein (GFP) in SD rats with middle cerebral artery occlusion (MCAO).and tO investigate the survival,migration and neural differentiation of the graft cells.Methods (1)CD34~+ cells were isolated from HUCB using centrifuge combined with immune beads and then identified by flow cytometry,and transfected by the recombinant plasmid of GDNF.GFP or GFP plasmid by liposome method.(2) Sixty aduh male SD rats with MCAO were randomly divided into three groups (n=20 in each group):GDNF-GFP-CD34~+ cells group,in which the GDNF-GFP-CD34~+ cells were transplanted intravenously at the 24th h after the establishment ofmodels of MCAO;GFP-CD34~+ cells group:in which the GFP-CD34~+ cells were transplanted intravenously at the Same time;Normal saline group,in which normal saline was injected at the same time.Fifteen SD rats served as sham-operated group.(3) Neurological functional measurements were performed using the modified neurolc'gical severity score.Quantitative histological determinations of infarct volume were performed using standard TTC staining and quantitative image analysis. The GDNF level in the cell culture or the cerebral tissue was measured by ELISA. Meanwhile, the survival and migration of GFP-labeled CD34~+ cells and the expression of astrocytie marker-GFAP and the neuron marker-neuronal nuclei (NeuN) were detected by immunohistochemical and fluorescent staining. Results (1) The GDNF level in the cell culture was significantly higher in GDNF-GFP-CD34~+ cells than in GFP-CD34~+ cells (P<0. 05). (2) No significant difference was found in the modified neurological severity score at the day 7 after transplantation among the three groups with MACO, but at day the 28 after transplantation, the neurological function in GFP-GDNF-CD34~+ group was improved significantly (5.0±1. O) as compared with the GFP-CD34~+ cells (5. 9 + 1.4) or saline groups (7. 0±1.7) (P<0. 05). The cerebral infarct volume in GFP-GDNF-CD34~+ group (142±44mm~3) was significantly decreased as compared with the GFP-CD34~+ group (196±58 mm~3) (P<0. 05) or saline group (233<50 mm~3 ) (P<0. 01 ). The GDNF level in cerebral tissue in GFP-GDNF-CD34~+ group was significantly increased as compared with the GFP-CD34~+ group (P<0. 05) at the 28th day after treatment. At the 28th day after treatment, the NeuN positive cells (6.7±2.0), and GFAP positive ceils (14.1±3.3) in GFP-GDNF-CD34~+ group were significantly increased as compared with the GFP-CD34~+ group (P<0. 05), but there were no positive cells in sham-operationgroup. Conclusion Intravenous transplantation of GDNF gene-modified CD34~+ cells from human umbilical cord blood could improve the neurological function in rats with MCAO. The increased GDNF level in cerebral tissue was one of possible mechanisms responsible for the different improvements.