骨髓间质干细胞移植对大鼠脑缺血再灌注凋亡蛋白Caspase-3的影响

Effect of Bone Marrow Mesenchymal Stem Cells Transplantation on Caspase-3 Expression in Cerebral Ischemia in Rats

目的研究静脉移植骨髓间充质干细胞(MSCs)对脑缺血再灌注模型大鼠神经功能及凋亡相关蛋白caspase-3的影响。方法体外培养及扩增MSCs后,用绿色荧光染料羟基荧光素二醋酸盐琥珀酰亚胺脂(CFSE)标记,通过静脉途径移植给大脑中动脉缺血2 h再灌注的SD大鼠,按不同时间点取材,荧光显微镜观察BMSCs在脑内的分布,免疫组织化学染色及RT-PCR检测大鼠脑内caspase-3蛋白表达情况。结果移植组在移植后第6天神经功能明显好于对照组(P<0.05)。移植组移植后3、12、24、48、72 h caspase 3免疫组化阳性目标面密度分别为(1.34±0.31)%、(3.98±0.67)%、(5.58±0.92)%、(4.65±0.69)%、(3.51±0.63)%,对照组分别为(2.09±0.19)%、(5.23±0.30)%、(6.89±0.57)%、(5.93±0.56)%、(4.39±0.57)%,移植组和对照组比较均(P<0.05)。6h及7 d移植组caspase 3阳性目标面密度分别为(2.81±0.35)%、(1.64±0.29)%,与对照组(3.92±0.44)%,(2.29±0.21)%比较差异显著(P<0.01)。移植组相应时间点caspase-3的表达明显低于对照组(P<0.05,P<0.01);移植组大鼠缺血侧皮层的caspase-3 mRNA相对量明显低于对照组(P<0.01)。结论经静脉注射骨髓间充质干细胞可明显改善神经功能。其可能通过下调caspase-3表达方式对脑缺血再灌注损伤起保护作用。

Objective To observe the distribution of intravenously transplanted bone marrow mesenchymal stem cells （BMSCs）, and their effects on neurological functions and the expression of apoptosis-related caspase-3 protein in cerebral ischemic rats. Methods BMSCs from SD rats were cultivated and proliferated in vitro, labeled with fluorescent dye CFSE. SD rats were subjected to transient middle cerebral artery occlusion （MCAO） for 2 hours and reperfusion. The rats were killed at different time points. The distribution of BMSCs was examined with fluorescence microscopy and the effects on caspase-3 protein expression by immunohistochemistry and reverse transcription polymerase chain reaction （RT-PCR）. Results The neurological functions of transplantation group were significantly superior to those of control group on the day 6 after transplantation （ P 〈 0.05）. At hour 3, 12, 24, 48, 72 after transplantation, the immunohistochemically positive target area density of caspase-3 was （ 1.34 ± 0.31 ） %,（3.98 ± 0.67） % , （5.58 ± 0.92） % , （4.65 ± 0.69） % , and （3.51 ± 0.63） % , respectively. Those of the control group were （2.09± 0.19）%, （5.23 ± 0.30）%, （6.89 ± 0.57）%, （5.93 ± 0.56）%, and （4.39 ± 0.57）%, respectively. The transplantation group was significantly superior to those of control group （ P 〈 0.05 ） ; At the hour 6 and on the day 7 after transplantation, the positive target area density of caspase-3 was （2.81 ± 0.35） % and （ 1.64 ± 0.29 ） %, respectively, those of the control group were （3.92 ± 0.44）% and （2.29 ± 0.21 ）%, respectively, the transplantation group was very significantly better than those of control group （ P 〈 0.01） ; The expression of caspase-3 was significantly reduced at different time points （ P 〈 0.05, P 〈 0.01 ）. The relative content of caspase-3 mRNA from rat ischemie cortex of transplantation group was very significantly lower than those of control group （ P 〈 0.01 ）. Conclusion Intravemous transplantation of bone marrow mesenebymal stem cells can improve the neurological functions. It is probably that the bone marrow mesenehymal stem cells exert protective effects on cerebral ischemia and reperfusion injury by down-regulating caspase-3 expression.