TGFβ1对MSCs体外模拟缺血性损伤的保护作用.docVIP

TGF-β1对MSCs体外模拟缺血性损伤的保护作用 　　作者：郑丽芳 作者单位：华中科技大学同济医学院附属协和医院神经内科,湖北 武汉 430022 　　【摘要】 目的 观察转化生长因子Β1(TGF-Β1)对骨髓间充质干细胞(MSCs)体外模拟缺血性损伤的保护作用。方法 采用密度梯度离心结合贴壁法分离、培养大鼠MSCs,对培养第3代的MSCs进行鉴定;用1、2、5 Μg/L 的TGF-Β1分别预处理细胞8 h,然后用血清剥夺和低氧(SOD)处理建立MSCs体外模拟缺血性损伤模型,通过流式细胞术评价MSCs的凋亡及存活情况;用Western blot方法检测2 Μg/L的TGF-Β1对Akt和p-Akt表达的影响。结果 密度梯度离心结合贴壁法能有效分离、纯化大鼠MSCs,免疫荧光检测显示培养的细胞CD29表达阳性,而CD34表达阴性。用2 Μg/L的TGF-Β1预处理MSCs后,可抑制SOD诱导的细胞凋亡,其早期凋亡率和中晚期凋亡率均明显下降,与凋亡模型组相比差异具有显著性(F=199.77、232.82,P0.05),而PI3K抑制剂Wortmannin能明显抑制TGF-Β1的抗凋亡作用。2 Μg/L的TGF-Β1可升高p-Akt的表达,p-Akt在8 h达到高峰,p-Akt/Akt显著增高,和其他时间点相比差异具有显著性(F=6.306,P0.05)。结论 TGF-Β1对SOD诱导MSCs的凋亡具有抑制作用,其抗凋亡作用是通过激活PI3K/Akt实现的。 　　【关键词】 转化生长因子Β 骨髓 间质干细胞 缺血 细胞保护 　　THE PROTECTIVE EFFECT OF TGF-Β1 TREATMENT ON MESENCHYMAL STEM CELLS INJURY CAUSED BY ANALOGOUS ISCHEMIA IN VITRO ZHENG LI-FANG, MEI YUANG-WU, ZHANG XIAO-QIAO, et al(Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China) [ABSTRACT]ObjectiveTo investigate the protective effect of TGF-Β1 on mesenchymal stem cells (MSCs) injury caused by analogous ischemia in vitro.MethodsDensity gradient centrifugalization (DGC) combined with adherence method were used to segregate and cultivate rat MSCs. MSCs cultivated to the 3 rd passage were characterized using flow cytometry technique. TGF-Β1 with 1,2 and 5 Μg/L were pretreated on MSCs for 8 h, then serum and oxygen deprivation were used to cause analogous ischemia of MSCs in vitro. AnnexinV/PI flow cytometry technique was used to evaluate apoptosis and survival of MSCs. Western blot technique was used to detect the expression of Akt and p-Akt when 2 Μg/L TGF-Β1 was pretreated on MSCs.ResultsDGC combined with adherence method could segregate and purify rat MSCs effectively. The results of flow cytometry showed that CD29 expression was positive while CD34 expression negative for MSCs. The results of AnnexinV/PI flow cytometry showed the number of apoptosis cell of 2 Μg/L TGF-Β1 pre