论文部分内容阅读
目的探讨转化生长因子-β1(TGF-β1)与丹参酮ⅡA(tanshinoneⅡA)联合诱导对大鼠骨髓间充质干细胞(BMSCs)定向分化为心肌样细胞的影响。方法采用全骨髓贴壁法从10只SD大鼠四肢骨中分离、培养BMSCs,流式细胞术对培养的细胞进行鉴定。对第2代BMSCs作定向诱导,根据加入诱导剂的不同分为TGF-β1组、tanshinoneⅡA组、两者联合诱导组及实验对照组(不加任何诱导剂)。诱导72h后更换为常规培养基继续培养,相差显微镜观察培养细胞的形态学变化;各组培养4周后,应用免疫细胞化学染色法检测原肌球蛋白(TPM)、缝隙连接蛋白43(Cx43)以及心肌肌钙蛋白I(c Tn I)的表达情况;实时定量PCR(Real-time PCR)法检测各组BMSCs在诱导培养的第1、2、4周心肌早期转录因子GATA-4、Nkx2.5的表达情况;透射电子显微镜观察分化细胞的超微结构变化。结果实验对照组细胞TPM、Cx43及c Tn I均为弱阳性或阴性表达。与实验对照组相比,TGF-β1组、TanshinoneⅡA组及两者联合诱导组BMSCs以上各标记物的阳性表达均明显升高,差异均具有统计学意义(均P<0.05)。其中,两者联合诱导组各标记物的阳性表达均显著高于TGF-β1及TanshinoneⅡA单独诱导组。Real-time PCR结果显示,在诱导第1周时TGF-β1组、TanshinoneⅡA组及两者联合诱导组GATA-4及Nkx2.5基因表达均最强,随后表达减弱至不表达。1周时,联合诱导组GATA-4 mRNA相对表达量是实验对照组的3.7倍,Nkx2.5 mRNA相对表达量是实验对照组的2.9倍,差异均具有统计学意义(均P<0.05)。透射电子显微镜结果显示,各诱导组均可见分化的细胞呈杆状,胞核卵圆形,位于细胞中央,胞质中可见平行排列的肌丝、粗面内质网和线粒体等细胞器。结论 TGF-β1、tanshinoneⅡA均可分别及联合诱导BMSCs获得心肌分化表型,且两者联合诱导效果优于单一诱导。
Objective To investigate the effects of TGF-β1 and tanshinoneⅡA on the differentiation of rat bone marrow-derived mesenchymal stem cells (BMSCs) into cardiomyocyte-like cells. Methods BMSCs were isolated from the bone of 10 SD rats using whole bone marrow adherent method. The cultured cells were identified by flow cytometry. The second generation of BMSCs were induced by induction, and divided into TGF-β1 group and tanshinoneⅡA group according to the different inducing agents. The combination of the two groups and experimental control group (without any inducer). After induction for 72 hours, the cells were replaced with conventional medium for further culture. The morphological changes of cultured cells were observed under phase contrast microscope. After cultured for 4 weeks, the expressions of tropomyosin (TPM), connexin 43 (Cx43) And cardiac troponin I (cTn I). Real-time quantitative PCR (Real-time PCR) was used to detect the expression of GATA-4 and Nkx2 on the 1st, 2nd, 4th week after induction of BMSCs. 5 expression; transmission electron microscopy of differentiated cells ultrastructure changes. Results The experimental control group cells TPM, Cx43 and c Tn I were weakly positive or negative expression. Compared with the experimental group, the positive expression of BMSCs in TGF-β1 group, TanshinoneⅡA group and the combination of the two groups were significantly increased (all P <0.05). Among them, the positive expression of each marker in both groups was significantly higher than that in TGF-β1 and TanshinoneⅡA alone groups. The results of Real-time PCR showed that the expression of GATA-4 and Nkx2.5 genes in TGF-β1 group, TanshinoneⅡA group and their combination induction group were strongest at the first week after induction, and then decreased to non-expression. At 1 week, the relative expression of GATA-4 mRNA in the co-induced group was 3.7 times that of the experimental group and the relative expression level of Nkx2.5 mRNA was 2.9 times that of the experimental group (all P <0.05). Transmission electron microscopy results showed that the differentiated cells were rod-shaped and oval nucleus in each group, located in the center of the cell. The myofilaments, rough endoplasmic reticulum and mitochondria and other organelles were arranged in parallel in the cytoplasm. Conclusion Both TGF-β1 and tanshinoneⅡA can induce cardiomyocyte differentiation phenotype respectively and in combination with BMSCs induction, and the combined effect of both is better than single induction.