丁酸钠和5-aza诱导间充质干细胞向心肌细胞分化的研究

目的探讨组蛋白去乙酰化酶抑制剂丁酸钠（sodiumbutyrate）和DNA甲基化抑制剂5-aza对骨髓间充质干细胞（BMSCs）向心肌细胞分化的影响。方法常规培养大鼠BMSCs，细胞分为4组：依次加入丁酸钠0mM，0.5mM，1.0raM，2.0mM。MTT和流式细胞仪检测不同浓度丁酸钠对细胞增殖和凋亡的影响。4周后利用western blot和免疫荧光检测4组细胞的心肌细胞特异性蛋白的表达。从上述4组中选出诱导能力最强的丁酸钠组与5-aza组成4个实验组：阴性对照组，5-aza组，丁酸钠组，5-aza.L丁酸钠组。作用4周后检测心肌细胞特异性蛋白的表达。结果丁酸钠抑制细胞增殖，具有浓度依赖性，而对细胞凋亡没有明显影响。检测发现丁酸钠和5-a．za均能有效诱导BMSCs分化为心肌细胞，丁酸钠诱导分化最适浓度为1.0mM。同时1．0mM丁酸钠诱导分化效率高于5-aza。除此，5-aza和丁酸钠共同作用于BMSCs，其诱导分化能力明显高于其它组。结论丁酸钠能够有效诱导BMSCs向心肌细胞分化，丁酸钠浓度为1．0mM时诱导能力最强，同时诱导分化率高于5-aza。5-aza和丁酸钠一起作用诱导BMSCs向心肌细胞分化的能力明显高于其它组，间接证明DNA去甲基化和组蛋白乙酰化具有协同作用。丁酸钠在有效作用浓度范围内，虽然抑制BMSCs增殖，但是不影响细胞凋亡，表明丁酸钠具有低毒的特性，为以后丁酸钠应用于临床打下实验基础。

Effects of sodium butyrate and 5-aza on differentiation of mesenchymal stem cells into cardiomyocytes

Objective To investigate the effects of sodium butyrate（histone deaeetylate inhibitor） and 5-aza（DNA demethylation inhibitor） on differentiation of rat bone marrow mesenehymal stem cells（BMSCs） into eardiomyocytes in vitro. Methods Rat bone marrow mesenchymal stem cells were isolated, cultured and identified. The second passage cells were divided into 4 groups,the concentration of sodium butyrate is 0mM 0.SmM, 1.0mM,2.0mM in each group respectively. Then the proliferation and apoptosis of the four groups were detected by MTT and flow cytometry.To study myocardial specific proteins in these groups after induction, the result was confirmed by immunofluorescenee and western blot.Then it studied the other four groups：the control group,5-aza,sodium butyrate,5-aza＋butyrate.the myocardial specific protein was identified after four weeks. Resuits sodium butyrate inhibited cell proliferation at all tested concentrations in a dose-dependent manner.However, it had no effect on cell apoptosis. sodium butyrate and 5-aza could induce BMSCs into cardiomyoeytes,The best effective concentration of sodium butyrate is 1 mM,and it is more prone to induce the differentiation of BMSCs than 5-aza.In additon,it is the most effective in differentiation with 5-aza and butyrate. Conclusion Sodium butyrate can promote the differentiation of BMSCs into eardiomyocytes effectively, and it is more prone than 5-aza with sodium butyrate at lmM. Besides,the co-culture group with 5-aza and sodium butyrate is the strongest capacity of cardiomyocyte-like cells differentiation,which shows a crosstalk between histone acctylation and DNA demethylation.In a effective induced range of concentration, sodium butyrate inhibited cell proliferation ,but it had rarely effect on apoptosis of BMSCs,its showed that it is low toxicity, which may lay the foundation for future clinical applications.