丁酸钠对大鼠骨髓间充质干细胞向平滑肌细胞分化的影响

Effects of sodium butyrate on differentiation of rat bone marrow mesenchymal stem cells into smooth muscle cells in vitro

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 effects of sodium butyrate,a histone deacetylase inhibitor（HDACi）,on differentiation of rat bone marrow mesenchymal stem cells（BMSCs） into smooth muscle cells in vitro.Methods Rat BMSCs and bladder smooth muscle cells（BSMCs） were isolated,identified,cultured to the third passage,and subjected to contact co-culture on two surfaces of a porous membrane.The co-culture medium for BMSCs contained 3.0 mmol/L sodium butyrate in an experimental group and 0 mmol/L sodium butyrate in a control group.The expression of smooth muscle markers,α-smooth muscle actin（α-SMA）,calponin,and smooth muscle-myosin heavy chain（SM-MHC）,was evaluated by RT-PCR and real-time PCR.The concentration of calponin in BMSCs was detected by immunofluorescence staining.Results The isolated rat BMSCs had positive expression of CD29,CD44,and CD166,with positive rates of 96.55%,92.76%,and 99.54%,respectively,and negative expression of CD31 and CD45,with positive rates of 1.39% and 5.56%,respectively.The isolated rat BSMCs had positive expression of α-SMA,calponin,and SM-MHC.The immunofluorescence staining results indicated that the expression of calponin increased gradually in both groups and was significantly higher in the experimental group.The RT-PCR and real-time PCR results showed that the gene expression levels of α-SMA,calponin,and SM-MHC increased gradually in both groups,with more obvious increase in the experimental group（P0.05）.Conclusion HDACi sodium butyrate can promote the differentiation of BMSCs into smooth muscle cells,suggesting that increasing the degree of histone acetylation can promote the differentiation of mesenchymal stem cells.