外源性和内源性凋亡通路在骨髓间充质干细胞移植修复大鼠脊髓损伤中的作用

Role of extrinsic and intrinsic apoptotic pathway in bone marrow stromal stem cell transplantation for spinal cord injury in rats

目的探讨骨髓间充质干细胞(BMSCs)移植修复大鼠脊髓损伤过程中外源性和内源性凋亡通路的作用。方法分离并扩增SD大鼠的BMSCs,以携带绿色荧光蛋白(GFP)基因的腺病毒转染BMSCs。采用Allen法构建脊髓损伤大鼠模型,并将30只脊髓损伤SD大鼠按照随机数字表法分为两组,每组15只。实验组:将携带GFP基因的BMSCs注入大鼠脊髓损伤区域附近。对照组:以无菌生理盐水注射于大鼠脊髓损伤区域附近。BMSCs移植1,2,3周后通过荧光显微镜观察携带GFP基因BMSCs的增殖情况,通过BBB评分观察大鼠行为能力变化,并采用TUNEL法观察脊髓损伤区域神经元凋亡状况。RT-PCR对B细胞淋巴瘤/白血病-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)、CD95特异性配体(FasL)、天冬氨酸蛋白水解酶-8(caspase-8)基因的表达进行分析。Western blot检测caspase-8蛋白的表达情况。结果携带GFP基因的BMSCs在脊髓损伤区域增殖。实验组BBB评分第2,3周时分别为(9.3±1.2)分、(12.5±2.2)分,较对照组高[分别为(5.2±1.1)分、(5.6±1.1)分](P<0.05或0.01);实验组1,2,3周的BBB评分逐渐升高(P<0.01)。实验组凋亡神经元数目在第2,3周时分别为(23.9±1.7)个、(10.5±1.9)个,均显著低于对照组[分别为(40.3±2.0)个、(37.2±2.6)个](P<0.01);实验组凋亡神经元在1,2,3周逐渐减少(P<0.01)。RT-PCR结果表明,实验组在第2,3周时Bcl-2的表达水平分别为0.50±0.02、0.71±0.04,均显著高于对照组(分别为0.23±0.02、0.21±0.01)(P<0.01);实验组Bcl-2的表达水平在第1,2,3周逐渐升高(P<0.01)。实验组第2,3周时Bax的表达水平分别为0.31±0.02、0.33±0.03,低于对照组(分别为0.52±0.06、0.78±0.04)(P<0.05或0.01);实验组Bax的表达在第1,2,3周差异无统计学意义(P>0.05),对照组第1,2,3周的Bax的表达水平逐渐升高(P<0.05)。实验组第2,3周Bcl-2/Bax的比值分别为1.62±0.06、2.16±0.27,显著高于对照组(分别为0.44±0.03、0.28±0.02)(P<0.01)。实验组第2,3周FasL的表达水平分别为0.42±0.04、0.27±0.02,低于对照组(分别为0.62±0.04、0.60±0.04)(P<0.05);实验组第1,2,3周FasL的表达水平逐渐降低(P<0.05)。实验组第2,3周时caspase-8基因的表达水平分别为0.38±0.01、0.16±0.02,低于对照组(分别为0.57±0.02、0.28±0.02)(P<0.05或0.01)。Western blot结果表明,实验组第2,3周时caspase-8蛋白的表达水平分别为0.28±0.06、0.26±0.05,低于对照组(分别为0.47±0.08、0.86±0.09)(P<0.05或0.01);实验组caspase-8蛋白的表达在第1,2,3周差异无统计学意义(P>0.05),对照组caspase-8蛋白的表达在1,2,3周逐渐升高(P<0.01)。结论BMSCs移植至脊髓损伤区后可显著改善脊髓损伤大鼠的行为能力,而这与其增强受损脊髓组织Bcl-2的表达,下调Bax、FasL、caspase-8的表达,抑制外源性和内源性凋亡程序从而减少神经凋亡有关。

Objective To investigate the role of extrinsic apoptotice pathway in transplanting bone.marrow stromal stem cells(BMSCs)for repair of spinal cord injury in rats.Methods BMSCs were isolated and amplifed from SD rats and were transfected with the adenovirus carrying the green fluorescent protein(CFP)gene.The Allen's method was used to construct the rat model of spinal cord injury.Thirty spinal cord injury rats were divided into two groups according to the random number table,with 15 rats per group.Around the spinal cord injury area,BMSCs carrying green fluorescent protein gene(GFP)were injected in experiment group,while sterile normal saline was injected in control group.After transplantation for 1,2 and 3 weeks,the proliferation of BMSCs was observed by fluorescence microscope,and the rats’behaviors were also assessed by BBB scale.The apoptosis of neurons in the spinal cord injury area was observed by TUNEL method.The mRNA expressions of B cell lymphoma/leukemia-2(Bcl-2),Bel-2 associated X protein(Bax),factor associated suicide ligand(FasL)and caspase-8 were determined by RT-PCR.The expression of caspase-8 was analyzed by Western blot method.Results The BMSCs carrying GFP genes proliferated in the spinal cord injury area.The BBB score in experiment group at2 and 3 weeks was(9.3±1.2)points and(12.5±2.2)points,higher than that in control group[(5.2±1.1)points,(5.6±1.1)points](P<0.05 or0.01).The BBB score in experiment group was gradually increased at 1,2,3 weeks(P<0.01).The number of apoptotic neurons in experiment group at 2 and 3 weeks was 23.9±1.7 and 10.5±1.9,significantly lower than that in control group(40.3±2.0,37.2±2.6)(P<0.01).The number of apoptotie neurons in experiment group showed a decreasing trend at 1,2,3 weeks(P<0.01).In RT-PCR analysis,the expression of Bcl-2 in experiment group at 2 and 3 weeks was 0.50±0.02 and 0.71±0.04,significantly higher than that in control group(0.23±0.02,0.21±0.01)(P<0.01).The expression of Bcl-2 in experiment group gradually increased at 1,2 and3 weeks(P<0.01).The expression of Bax in experiment group at 2 and 3 weeks was 0.31±0.02 and 0.33±0.03,lower than that in control group(0.52±O.06,0.78±0.04)(P<0.05 or0.01).The expression of Bax in experiment group showed no significant difference at 1,2,and3 weeks(P>0.05).The expression of Bax in control group gradually increased at 1,2 and 3 weeks(P<0.05).The ratio of Bcl-2/Bax in experiment group was I.62±0.06 and 2.16±0.27,significantly higher than that in control group(0.44±0.03,0.28±0.02)(P<0.01).The expression of FasL in experiment group at 2,3 weeks was 0.42±0.04 and 0.27±0.02,lower than that in control group(0.62±0.04,0.60±0.04)(P<0.05).The expression of FasL in experiment group was gradually decreased at 1,2 and3 weeks(P<0.05).The expression of caspase-8 in experiment group was 0.38±0.01 and 0.16±0.02,lower than that in control group(0.57±0.02,0.28±0.02)(P<0.05 or0.01).In Western blot analysis,the protein expression of caspase-8 in experimental group at 2,3 weeks was0.28±0.06 and 0.26±0.05,lower than that in control group(0.47±0.08,0.86±0.09)(P<0.05 or0.01).There was no statistically significant difference in the protein expression of Caspase-8 protein in experimental groupat 1,2,and 3 weeks(P>0.05).The protein expression of caspase-8 in control group gradually increased at 1,2,and 3 weeks(P<0.05).Conclusion Transplantation of BMSCs to the spinal cord injury area can significantly improve the behavioral performance of rats.This is correlated with reduced neuronal apoptosis by up-regulation of Bel-2 expression and down-regulation of Bax,FasL and caspase-8 expression to inhibit extrinsic and intrinsic apoptotic pathway.