组蛋白乙酰化修饰在大鼠bmscs向bsmcs分化中 - 第三军医大学学报.doc

大鼠BMSCs向BSMCs分化的标志基因表达受组蛋白乙酰化调控(400038重庆，第三军医大学西南医院妇产科)[摘要] 目的 通过骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)与膀胱平滑肌细胞(bladder smooth muscle cells, BSMCs)接触共培养探讨组蛋白乙酰化对BMSCs分化能力的影响。方法　分别采用密度梯度离心法和胶原酶消化法培养大鼠BMSCs及BSMCs至第3代，并对BMSCs进行了鉴定；以单独培养的BMSCs作为对照组，接触共培养的BMSCs为实验组；运用免疫荧光化学染色法分别对2组BMSCs中的平滑肌标志性蛋白平滑肌α肌动蛋白(α-smooth muscle actin, α-SMA)、平滑肌肌球蛋白重链(smooth muscle-myosin heavy chain, SM-MHC)、Calponin进行检测，estern blot检测α-SMA蛋白、Calponin蛋白在BMSCs中表达并用微球菌核酸酶(Micrococcal Nuclease MNase)对平滑肌三个标志性基因的启动子区进行了染色质开放性的检测，同时运用了ChIP技术分析了α-SMA、SM-MHC启动子区组蛋白乙酰化水平对BMSCs向平滑肌分化的影响。结果　大鼠原代BMSCs流式检测显示CD29表达阳性,阳性率为99.8%CD31、CD45表达阴性，阳性率为6.30%、1.04%；免疫荧光显示平滑肌3种标志性蛋白的表达均随时间的延长而增加；Western Blot结果显示随着共培养天数的增加α-SMA蛋白、Calponin蛋白在实验组中的表达相比对照组有明显的增加MNase显示实验组的BMSCs启动子区染色质对核酸酶的敏感性明显高于对照组，ChIP显示实验组中BMSCs平滑肌标志性基因α-SMA、SM-MH启动子区H4乙酰化水平较未分化前明显增加(P0.05)。结论 组蛋白乙酰化程度的增加促进了BMSCs向BSMCs分化。 [关键词]　骨髓间充质干细胞膀胱平滑肌细胞组蛋白乙酰化启动子histone acetylation on the expression of marker genes in differentiation of rat BMSCs to BSMCs Xu Mengyao, WangYanzhou, Xu Huicheng (Department of Gynecology and Obstetrics, Southwest Hospital, Thrid Military Medical University, Chongqing, 400038, China) [Abstract] Objective Mesenchymal stem cells in the bone marrow (bone marrow mesenchymal stem cells, BMSCs) and bladder smooth muscle cells (bladder smoothmuscle cells, BSMCs) coculture histone acetylation BMSCs differentiation capacity. Methods Density gradient centrifugtion and collagenase digestion method were used to train the rats BMSCs and BSMCs to the 3rd generation, and identify the BMSCs. BMSCs cultured alone as the control group, which contacts the co-cultured BMSCs for the experimental group. Using immune fluorescence staining on the smooth muscle of the two groups which is BMSCs landmark protein on the smooth muscle α actin (α-smooth muscle actin, α-SMA) and smooth muscle myosin heavy chain (smooth muscle-myosin heavy chain, SM-MHC) which will be detected. Western Blot was adopted to detect the level of α-SMA and Calponin protein in