摘要
目的探讨丹酚酸B联合诱导多能干细胞来源的神经干细胞（iPSCs-NSCs）对大鼠脑缺血再灌注损伤后神经功能的修复作用，以及对基质金属蛋白酶-2（MMP-2）及其抑制剂金属蛋白酶组织抑制因子-2（TIMP-2）表达的影响。并观察蛋白激酶B（Akt）信号传导通路在该过程中的作用。方法将诱导多能干细胞（iPSCs）诱导分化为神经干细胞（NSCs），并行巢蛋白（nestin）免疫荧光染色。（1）其后进一步诱导分化为神经元，并分为4组：常规培养基组、丹酚酸B组（加入丹酚酸B50μmol／L）、丹酚酸B＋GM6001组（加入丹酚酸B50μmol／L、GM6001 25μmol／L）、丹酚酸B＋LY294002组（加入丹酚酸B50μmol／L、LY294002 25μmol／L）。分化后细胞进行微管相关蛋白2（MAY2）免疫荧光染色和实时定量PCR检测，并采用Western blotting检测常规培养基组、丹酚酸B组、丹酚酸B＋LY294002组MMP-2、pAkt和Akt表达。（2）48只大脑中动脉闭塞模型大鼠按随机数字表法分为3组：PBS空白组、iPSCs-NSCs组、丹酚酸B＋iPSCs-NSCs组，分别于纹状体处局部注射PBS、iPSCs-NSCs及丹酚酸B。造模7、14d后应用Longa评分和SMA评分对各组大鼠神经功能进行评估：7d后采用免疫荧光染色检测nestin表达，14d后采用免疫荧光染色检测MAP2、胶质纤维酸性蛋白（GFAP）表达；采用Western blotting检测各组MMP-2、TIMP-2表达。结果（1）与常规培养基组、丹酚酸B＋GM6001组、丹酚酸B＋LY294002组比较，丹酚酸B组神经元计数、MAP2 mRNA相对表达量明显增高，差异均有统计学意义（P〈0．05）。与常规培养基组、丹酚酸B＋LY294002组比较，丹酚酸B组MMP-2、pAkt／Akt表达明显增高，差异均有统计学意义（P〈0．05）。（2）造模7、14d后，与PBS空白组、iPSCs-NSCs组比较。丹酚酸B＋iPSCs-NSCs组Longa评分、SMA评分明显降低，差异均有统计学意义（P〈0．05）；14d后，与iPSCs-NSCs组
Objective To study the effects of Salvianolic acid B （Sal B） induced pluripotent stem cells-derived neural stem cells （iPSCs-NSCs） on restoration of nerve function, expressions of matrix metalloproteinase-2 （MMP-2） and tissue inhibitor of metalloproteinase-2 （TIMP-2） in rats after ischemia reperfusion injury, and explore the role of protein kinase B （PKB/Akt） signal pathway in these processes. Methods The iPSCs-NSCs were induced and differentiated into NSCs, and immunofluorescent staining was performed to detect the nestin expression in the cells. （1） NSCs were further induced into neurons; these neurons were divided into routine medium group, Sal B group （adding 50 μmol/L Sal B）, Sal＋GM6001 group （adding 50μmol/L Sal B＋25 μmol/L GM6001）, and Sal B＋LY294002 group （adding 50 μmol/L Sal B＋25 μmol/L LY294002）. The cells were counted under microscope, and microtubule-associated protein （MAP） 2 expressions were detected by immunofluorescent staining and real time PCR. The protein levels of matrix metalloprotein （MMP）-2, phosphorylated （p-） Akt and Akt were detected by Western blotting. （2） Forty-eight SD rats with middle cerebral artery occlusion （MCAO） were randomly divided into 3 groups： PBS blank group （giving PBS）, iPSCs-NSCs group （giving PSCs-NSCs）, Sal B＋iPSCs-NSCs group （giving Sal B＋iPSCs-NSCs）; local injection was performed into the corpus striatum. Seven and 14 d after that, Longa grading and SMA grading were performed to evaluate the neurological functions; 7 d after that, immunofluorescent staining was performed to detect the nestin expression; 14 d after that, immunofluorescent staining was performed to detect the MAP2 and glial fibriUary acidic protein （GFAP） expressions. Western blotting was employed to detect the MMP-2 and TIMP-2 protein expressions. Results （1） In vitro, the number of neurons and MAP2 mRNA expression level in Sal B group were significantly larger/higher as compared with those in routine med
出处
《中华神经医学杂志》
CAS
CSCD
北大核心
2016年第11期1084-1090,共7页
Chinese Journal of Neuromedicine
基金
国家自然科学基金（31170947）
中国博士后科学基金（2014M552272）
