Janus激酶信号转导及转录激活因子通路磷酸化水平在干细胞移植治疗大鼠局灶脑缺血/再灌注神经损伤中的作用

Role of phosphorylation of janus kinase-signal transducer and activators of transcription 3 signaling pathway in stem cell transplantation for the treatment of focal cerebral ischemia/reperfusion nerve injury in rats

目的探讨Janus激酶信号转导及转录激活因子(JAK-STAT)信号通路在Neurogin2基因(Ngn2)诱导的神经潜能骨髓间充质干细胞(Ngn2-MSCs)移植治疗大鼠局灶性脑缺血/再灌注(MCAO)损伤中的作用。方法体外培养大鼠MSCs细胞, 并将Ngn2基因通过慢病毒转染MSCs细胞;20只SD大鼠按照随机数字表法分为两组, 制备大鼠MCAO模型, 分别设为Ngn2-MSCs移植组、Ngn2-MSCs+JAK-STAT通路抑制剂(AG490)移植组。干细胞移植后0、12、24、48、60、72、90 h进行神经缺陷症状评分单因素方差分析, 蛋白质印迹法(Western blot)检测STAT3及磷酸化STAT3蛋白表达水平, 并采用两均数成组设计资料t检验分析。原位缺口末端标记法(TUNEL)法检测脑缺血再灌注区细胞凋亡水平并采用单因素方差分析。结果 Ngn2转染MSCs后, Ngn2-MSCs细胞表现出向神经细胞分化的潜能, Western blot检测提示干细胞移植后脑组织JAK-STAT3信号通路12 h开始STAT3蛋白磷酸化激活, 48 h达到高峰, 90 h后逐渐下降, 均高于0 h(12 h:t=4.780, 48 h:t=6.991, 90 h:t=4.578, P均<0.05)。实验组细胞凋亡数量48 h[(20.5±2.1)个/高倍镜视野]及其他时间点均低于对照组(组间F=13.372, P<0.05), 神经缺陷评分(2.51±0.16 48 h及之后时间点)均低于对照组(组间F=6.990, P<0.05)。结论干细胞移植治疗大鼠脑缺损损伤中, JAK-STAT3通路磷酸化激活与神经损伤相关, 抑制JAK-STAT3通路磷酸化水平可以降低细胞移植区细胞凋亡水平, 提高干细胞移植存活率, 从而起到神经保护作用。

Objective To investigate the role of janus kinase-signal transducer and activators of transcription 3(JAK-STAT3)signaling pathway in the treatment of focal cerebral ischemia/reperfusion injury in rats by Neurogin2(Ngn2)-induced neural-potential bone marrow mesenchymal stem cells(Ngn2-MSCs)transplantation.Methods Rat bone marrow mesenchymal stem cells(MSCs)were cultured in vitro and transfected with Ngn2 gene by lentivirus.The rat middle cerebral artery embolism(MCAO)model was established.According to random number table,the animals were divided into two groups:Ngn2-MSCs transplantation group and Ngn2-MSCs+JAK-STAT3 signaling pathway inhibitor(AG490)transplantation group.Neurological defect symptom score was recorded at 0,12,24,48,60,72 and 90 h after stem cell transplantation and analysed by one-way ANOVA.STAT3 and phosphorylated STAT3 protein expression levels were detected by Western blotting,protein gray scale was detected compared with internal reference gray scale,and apoptosis levels of cerebral ischemia reperfusion area were detected by terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL)assay,and the mean number of cells was analyzed by one-way ANOVA.Results After Ngn2 transfection of MSCs,Ngn2-MSCs showed the potential to differentiate into nerve cells.Western blotting analysis showed that the JAK-STAT3 signaling pathway in brain tissue began to phosphorylation activation of STAT3 protein at 12 h after stem cell transplantation,and reached a peak at 48 h.After 90 h,it gradually decreased,and it gradually decreased and was higher than 0 h(12 h:t=4.780,48 h:t=6.991,90 h:t=4.578,P<0.05).Compared with the control group,the number of apoptotic cells in the experimental group decreased significantly at 48 h[(20.5±2.1)/high power field]and that at other time points was lower than that in the control group(inter-group F=13.372,P<0.05),and the neurological defect score(2.51±0.16 at 48 h and subsequent time points)was lower than that in the control group(inter-group F=6.990,P<0.05).Conclusion Phosphorylation activation of JAK-STAT3 pathway is related to nerve injury in the treatment of rat brain injury by stem cell transplantation.Inhibition of phosphorylation of JAK-STAT3 pathway can reduce the level of apoptosis,thus playing a neuroprotective role.