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目的探讨组蛋白去乙酰化酶抑制剂丁酸钠(sodium butyrate)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,1.0mM,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+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-aza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。
Objective To investigate the effects of sodium butyrate, a histone deacetylase inhibitor, and 5-aza, a DNA methylation inhibitor, on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into cardiomyocytes. Methods BMSCs were cultured routinely. The cells were divided into 4 groups: sodium butyrate 0mM, 0.5mM, 1.0mM, 2.0mM. The effects of different concentrations of sodium butyrate on cell proliferation and apoptosis were detected by MTT and flow cytometry. After 4 weeks, the expression of cardiomyocyte-specific protein in four groups of cells was detected by western blot and immunofluorescence. From the above four groups, 4 sodium butyrate group and 5-aza group were selected as the most inducing group to form four experimental groups: negative control group, 5-aza group, sodium butyrate group and 5-aza + sodium butyrate group. After 4 weeks, the expression of cardiomyocyte specific protein was detected. Results Sodium butyrate inhibited cell proliferation in a concentration-dependent manner, but had no obvious effect on apoptosis. Found that sodium butyrate and 5-aza can effectively induce the differentiation of BMSCs into cardiomyocytes, the optimum concentration of sodium butyrate induced differentiation 1.0mM. At the same time 1.0mM sodium butyrate induced differentiation efficiency higher than 5-aza. In addition, 5-aza and sodium butyrate co-act on BMSCs, their ability to induce differentiation was significantly higher than other groups. Conclusions Sodium butyrate can effectively induce the differentiation of BMSCs into cardiomyocytes. When sodium butyrate concentration is 1.0mM, the induction rate is the strongest and the differentiation rate is higher than that of 5-aza. 5-aza combined with sodium butyrate to induce BMSCs differentiate into cardiomyocytes significantly higher than the other groups, indirectly proving the synergistic effect of DNA demethylation and histone acetylation. Although sodium butyrate inhibited the proliferation of BMSCs in the range of effective concentration, it did not affect the apoptosis, indicating that sodium butyrate had the characteristics of low toxicity, which laid the experimental foundation for the future application of sodium butyrate in clinical practice.