丁酸钠协同成纤维细胞生长因子2体外诱导骨髓间充质干细胞向心肌细胞分化的研究

Study of sodium butyrate combined with fibroblast growth factor 2 on differentiation of bone marrow mesenchymal stem cells into cardiomyocytes

目的探讨丁酸钠协同成纤维细胞生长因子2(FGF-2)体外诱导骨髓间充质干细胞(BMMSC)向心肌细胞分化的可行性。方法经全骨髓贴壁法分离纯化BMMSC,进行流式细胞技术鉴定;分别加入普通完全培养基(对照组)、1 mmol/L丁酸钠培养基(丁酸钠组)、1 ng/ml FGF-2的培养基(FGF-2组)、1 mmol/L丁酸钠和1 ng/ml FGF-2的培养基(联合组)诱导培养。提取四组细胞加药诱导后1、2、4周的总RNA,通过定量反转录PCR(qRT-PCR)检测心肌特异性转录因子。运用免疫组织化学和Western blot技术,检测BMMSC诱导4周后心肌特异性蛋白的表达。结果原代细胞培养48 h后呈圆扁形、贴壁生长,有些细胞形态已出现短梭形改变;培养1周的BMMSC进一步伸展,出现梭形、多角形和不规则形等多种形态;诱导培养4周后的BMMSC,相邻细胞间具有明显的方向性排列,一些细胞甚至成了漩涡样结构。流式细胞仪结果证明分离的细胞为纯化的BMMSC。qRT-PCR检测结果:心肌特异性基因GATA-4在诱导2周表达最强,联合组明显高于其他各组(P<0.05)。Western blot和免疫细胞化学检测结果:BMMSC经培养4周后的心脏特异性蛋白联合组高于其他各组(P<0.05)。结论丁酸钠和FGF-2诱导BMMSC向心肌样细胞的分化,联合诱导比单独诱导效果好。

Objective To investigate the feasibility of inducing the differentiation of bone marrow mesenchymal stem cells(BMMSC)into cardiomyocytes by sodium butyrate and fibroblast growth factor 2(FGF-2)in vitro.Methods BMMSC were isolated and purified by whole bone marrow adherent method and identified by flow cytometry.Normal complete medium(control group),1 mmol/L sodium butyrate medium(sodium butyrate group),1 ng/ml FGF-2 medium(FGF-2 group),1 mmol/L sodium butyrate and 1 ng/ml FGF-2 medium(combined group)were added to induce culture.Total RNA was extracted from four groups at 1,2 and 4 weeks after induction.Myocardial specific transcription factors were detected by quantitative reverse transcriptase-mediated PCR(qRT-PCR).Immunocytochemistry and Western blot were used to detect the expression of myocardial specific proteins induced by BMMSC after four weeks.Results After 48 h of culture,the primary cells showed oblate and adherent growth,and some cells had short fusiform changes.BMMSC cultured for one week were further extended and showed spindle shape,polygon shape and irregular shape.BMMSC cultured for four weeks showed obvious directional arrangement between adjacent cells,and some cells even formed whirlpool-like structures.Flow cytometry results showed that the isolated cells were purified BMMSC.The results of qRT-PCR showed that the expression of myocardium specific gene GATA-4 was the highest after two weeks of culture,and the expression in the combined group was significantly higher than that in the other groups(P<0.05).The results of Western blot and immunocytochemistry showed that after four weeks of BMMSC culture,the heart-specific protein level in combined group was higher than those in the other groups(P<0.05).Conclusion Sodium butyrate and FGF-2 induced the differentiation of BMMSC into cardiomyoid cells,and the combined induction is better than the single induction.