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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,Western blot检测α-SMA蛋白、Calponin蛋白在BMSCs中的表达,并用微球菌核酸酶(micrococcal nuclease,MNase)对平滑肌3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-MHC、Calponin启动子区H4乙酰化水平较未分化前明显增加(P<0.05)。结论组蛋白乙酰化程度的增加促进了BMSCs向BSMCs分化。
Objective To investigate the effect of histone acetylation on differentiation of BMSCs by contact co-culture of bone marrow mesenchymal stem cells (BMSCs) and bladder smooth muscle cells (BSMCs). Methods BMSCs and BSMCs were cultured to the third passage of rat by density gradient centrifugation and collagenase digestion, and BMSCs were identified. BMSCs cultured alone were used as a control group and exposed to co-cultured BMSCs as experimental group. Immunization The expressions of smooth muscle-smooth muscle actin (α-SMA), smooth muscle-myosin heavy chain (SM-MHC) , Calponin. Western blot was used to detect the expression of α-SMA protein and Calponin protein in BMSCs. The promoter regions of three markers of smooth muscle were chromatin-opened with micrococcal nuclease (MNase) At the same time, ChIP was used to analyze the effects of α-SMA and SM-MHC promoter histone acetylation on smooth muscle differentiation of BMSCs. Results The flow cytometry of primary rat BMSCs showed that the expression of CD29 was positive, the positive rate was 99.8%, the expression of CD31 and CD45 was negative, the positive rate was 6.30% and 1.04% respectively. Immunofluorescence showed that the expression of three markers of smooth muscle Prolonged and increased; Western blot results showed that with the increase of co-culture days α-SMA protein Calponin protein expression in the experimental group compared with the control group increased significantly; MNase showed that the experimental group of BMSCs promoter region chromatin on the nucleic acid ChIP showed that the H4 acetylation level of α-SMA, SM-MHC and Calponin promoter regions in smooth muscle of BMSCs in experimental group was significantly higher than that in control group (P <0.05). Conclusion The increase of histone acetylation promotes the differentiation of BMSCs into BSMCs.