Bcl-2基因修饰的骨髓间充质干细胞移植治疗脑缺血的实验研究  

Therapeutic benefit of intravenous transplantation of Bcl-2 gene-modified MSCs after cerebral ischemia in rats

作　　者：张小乔[1] 梅元武[2] 郑丽芳[2] 张红[2] 郭远瑾[2] 徐江华[2] 

机构地区：[1]湖北省十堰市郧阳医学院附属太和医院干部科,442000 [2]华中科技大学同济医学院附属协和医院神经内科

出　　处：《卒中与神经疾病》2009年第5期270-275,共6页Stroke and Nervous Diseases

摘　　要：目的观察Bcl-2基因修饰的骨髓间充质干细胞(MSCs)移植对大鼠脑缺血性损伤的治疗作用及对移植的MSCs保护性作用。方法将114只SD大鼠随机分为假手术组、Model组、MSCs组和Bcl-2-MSCs组;线栓法制作大鼠一侧大脑中动脉缺血再灌注模型,MSCs组及Bcl-2-MSCs组在缺血24h后经尾静脉注射方式移植BrdU标记的MSCs及人Bcl-2基因修饰的MSCs;分别于术后1、7、14、28d对各组大鼠进行神经功能缺损评分(NSS);在脑缺血14d应用TTC法观察梗死灶体积;BrdU和TUNEL免疫荧光双重标记检测移植的MSCs凋亡情况;Westernblot检测大鼠脑梗死周边区Bcl-2蛋白的表达;HE染色观察脑组织病理形态。结果MSCs-Bcl-2组和MSCs组NSS评分、脑梗死体积百分比、TUNEL阳性细胞数较Model组低(P<0.05),且MSCs-Bcl-2组比MSCs组更低,BrdU阳性细胞数较多(P<0.05);MSCs-Bcl-2组BrdU和TUNEL免疫荧光双标细胞数稍多,但差异不显著(P>0.05),而MSCs-Bcl-2组BrdU和TUNEL免疫荧光双标细胞占BrdU阳性细胞百分比明显较低(P<0.05)。MSCs-Bcl-2组Bcl-2蛋白呈持续较高水平的表达,和MSCs组同时间点相比差异有统计学意义(P<0.05)。HE染色示MSCs组和MSCs-Bcl-2组脑组织损伤及细胞丢失较轻,MSCs-Bcl-2组更明显,脑梗死周边区均未见到核大、浓染的异形细胞。结论Bcl-2基因修饰的MSCs移植较MSCs移植能进一步改善脑缺血大鼠的神经功能,减少脑梗死体积;其机制为Bcl-2基因修饰使MSCs持续、稳定表达一定量的Bcl-2蛋白,从而能保护移植的MSCs,减少其凋亡,增加其存活。Objective To survey the therapeutic benefit of intravenous transplantation of Bcl-2 genemodified MSCs after ischemia in rats and the protective effect of Bcl-2 genemodification to transplanted MSCs. Methods The rat model of focal cerebral ischemia was established with unilateral middle cerebral artery suture occlusion method and reperfused 2h later with suture extracted. Rats in MSCs group and Bcl-2-MSCs group were transplanted MSCs and human Bcl-2 gene-modified MSCs labeled with 5-bromodeoxy-uridine （BrdU） respectively through tail vein intravenous injection. Neurological function of rats was evaluated with modified Neurological Severity Scores （NSS） after ischemia. Cerebral infarction volume of rats was observed through tetrazolium chloride （TTC） staining. BrdU and terminal deoxynucteotide transferase-mediated dUTP nick end-labeling （TUNEL） immunofluorescence double labeling technique was used to detect apoptosis of transplanted MSCs. Pathologic morphous of brain issue after ischemia was survey through HE staining. The expression of Bcl-2 protein in cortex around cerebral infarction was detected through Western blot. Results The NSS score, the percentage of cerebral infarction volume, the number of TUNEL positive cell in MSCs group and Bcl-2-MSCs group were lower than those of model group （P 〈 0. 05）. To compare with MSCs group, those indexes of Bcl-2-MSCs group were more lower （P〈0. 05）. The number of BrdU positive cell in Bcl-2-MSCs group was more than that of MSCs group （P〈0. 05）. The number of BrdU and TUNEL double labeling positive cell in Bcl-2-MSCs group was more than that of MSCs group but the difference was not significant（P〉0. 05）. On different time point after ischemia, the expression level of Bcl-2 protein was higher continuously in Bcl-2-MSCs group than that of MSCs group （P〈0. 05）. HE stain showed that the brain tissue damage was milder, the number of lost cell was fewer in Bcl-2-MSCs＇group and MSCs group than that of model group. There were not kary

关 键 词：BCL-2 基因修饰 骨髓间充质干细胞 移植 脑缺血 

分 类 号：R743[医药卫生—神经病学与精神病学]