MicroRNA-9调控室管膜下区干细胞增殖在电针治疗局灶性脑缺血中的作用

摘要

目的：探讨电针曲池、足三里穴是否是通过介导microRNA-9(miR-9)调控缺血再灌注损伤大鼠室管膜下区神经干细胞增殖来发挥神经保护作用。方法52只Sprague-Dawley大鼠随机分为假手术组(n=12)、模型组(n=12)、电针组(n=12)、电针+二甲基亚砜(DMSO)溶剂组(n=8)和电针+miR-9模拟物组(n=8)。制作大鼠大脑中动脉闭塞(MCAO)致局灶性脑缺血模型，电针+DMSO溶剂组和电针+miR-9模拟物组分别在造模前30 min侧脑室注射0.7%DMSO溶剂和miR-9模拟物。术后第2天电针患侧曲池、足三里穴。造模后分别在第2天至第6天腹腔注射溴脱氧尿苷(BrdU)；免疫荧光观察BrdU与巢蛋白(Nestin)共定位；RT-qPCR检测室管膜下区miR-9相对表达量。结果电针后7 d，电针组神经缺损功能评分明显低于模型组(t=9.600, P=0.006)，脑梗死体积小于模型组(t=14.080, P=0.024)。电针组室管膜下区Nestin以及BrdU与Nestin共定位阳性细胞数量均增加，同时室管膜下区miR-9低表达(P<0.05)。电针+miR-9模拟物组BrdU、Nestin阳性细胞数量以及BrdU与Nestin共定位的数量较电针+DMSO溶剂组减少。结论电针曲池、足三里穴可促进室管膜下区神经干细胞增殖，其作用可能通过下调miR-9表达。%Objective To investigate the effect of electroacupuncture (EA) at Quchi (LI11) and Zusanli (ST36) acupoints on rats with fo-cal cerebral ischemia, and whether it was mediated by microRNA-9 (miR-9) regulating neural stem cells proliferation in subventricular zone. Methods Fifty-two Sprague-Dawley rats were randomly assigned into sham group (n=12), model group (n=12), EA group (n=12), EA+dimethylsulfoxide (DMSO) group (n=8) and EA+miR-9 mimics group (n=8). The middle cerebral artery occlusion model was induced. DMSO and miR-9 mimics were performed by intracerebroventricular injection 30 minutes before modeling. The EA group, EA+ DMSO group and EA+miR-9 mimics group were electroacupunctured at Quchi and Zusanli acupoints the next day after modeling. All the groups were intraperitoneally injected with bromodeoxyuridine (BrdU). The BrdU co-localized with Nestin was observed by immunofluorescence. The expression of miR-9 in subventricular zone was detected by real time qPCR. Results The neurological deficit score was lower (t=9.600, P=0.006), and the infarct volume was smaller (t=14.080, P=0.024) in the electroacupuncture group than in the model group. The expression of Nestin and BrdU co-localizated with Nestin increased;while the expression of miR-9 decreased in subventricular zone (P<0.05) in the electoacupuncture group. However, the expression of Nestin, BrdU and BrdU co-localizated with Nestin was lower in the EA+miR-9 mim-ics group than in the EA+DMSO group. Conclusion Electroacupuncture at Quchi and Zusanli acupoints may promote the neural stem cells proliferation in subventricular zone by downregulating the miR-9 expression.