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目的探讨组蛋白去乙酰化酶抑制剂(histone deacetylase inhibitors,HDACi)丁酸钠对在体外与膀胱平滑肌细胞(bladder smooth muscle cells,BSMCs)接触共培养的骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)分化能力的影响。方法常规培养大鼠BMSCs及BSMCs至第3代,种植于多孔膜两侧进行接触共培养。实验组BMSCs培养上室面添加含3 mmol/L丁酸钠诱导其分化;对照组不含丁酸钠行接触共培养。免疫荧光化学染色法对2组BMSCs中平滑肌标志性蛋白calponin进行检测,同时用逆转录PCR(RT-PCR)和荧光定量PCR法(real-time PCR)对BMSCs中平滑肌α肌动蛋白(α-smooth muscle actin,α-SMA)、calponin、平滑肌肌球蛋白重链(smooth muscle-myosin heavy chain,SM-MHC)行进鉴定。结果大鼠原代BMSCs流式检测显示CD29、CD44、CD166表达阳性,阳性率分别为96.55%、92.76%、99.54%;CD31、CD45表达阴性,阳性率为1.39%、5.56%。BSMCs鉴定α-SMA、calponin、SM-MHC 3种标志性蛋白的表达均为阳性。细胞免疫荧光染色显示平滑肌标志性蛋白calponin的表达均随时间的延长而增加,实验组表达强度明显高于对照组。RT-PCR和荧光定量PCR显示实验组中BMSCs平滑肌标志性基因α-SMA、calponin、SM-MHC随共培养时间的延长逐渐增多,与对照组比较有明显差异(P<0.05)。结论 HDACi丁酸钠可以增强BMSCs向平滑肌细胞的分化能力,提示提高组蛋白乙酰化程度可以促进BMSCs分化。
Objective To investigate the effect of HDACi sodium butyrate on bone marrow mesenchymal stem cells (c-MSCs) co-cultured with bladder smooth muscle cells (BSMCs) in vitro. , BMSCs) differentiation ability. Methods BMSCs and BSMCs were cultured in the third generation in the conventional way and cultivated on both sides of the porous membrane for co-culture. Experimental group BMSCs cultured on the room to add 3 mmol / L sodium butyrate to induce differentiation; control group without sodium butyrate contact co-culture. Immunofluorescence staining was used to detect the smooth muscle marker calponin in two groups of BMSCs. Reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR (Real-time PCR) smooth muscle actin (α-SMA), calponin, smooth muscle-myosin heavy chain (SM-MHC). Results The flow cytometric analysis of primary BMSCs showed that the positive rates of CD29, CD44 and CD166 were 96.55%, 92.76% and 99.54%, respectively. The positive rates of CD31 and CD45 were 1.39% and 5.56% respectively. BSMCs identified positive expression of the three markers of a-SMA, calponin and SM-MHC. Immunofluorescence staining showed that the expression of calponin, a marker of smooth muscle, increased with time, and the expression intensity of the experimental group was significantly higher than that of the control group. RT-PCR and real-time PCR showed that the smooth muscle a-SMA, calponin and SM-MHC of BMSCs in experimental group increased gradually with the extension of co-culture time, which were significantly different from the control group (P <0.05). Conclusion HDACi sodium butyrate can enhance the differentiation of BMSCs into smooth muscle cells, suggesting that increasing histone acetylation may promote the differentiation of BMSCs.