内皮素-1促进兔骨髓间质干细胞诱导分化为心肌样细胞

Effect of endothelin-1 on differentiation of cardiomyogenic cells induced from rabbit bone mesenchymal stem cells

目的研究ET-1对兔骨髓间质干细胞(BMSCs)定向心肌样细胞分化的影响。方法取成年兔股骨干BMSCs培养。实验分为6组Ⅰ组,未诱导组;Ⅱ组,单纯ET-1组(30nmol/L);Ⅲ组,5-aza诱导组(10μmol/L);Ⅳ组,5-aza+ET-1联合诱导组。5-aza诱导3周后,加入ET-1。其中又分为Ⅳ1组、Ⅳ2组、Ⅳ3组,分别为加入10、30和50nmol/LET-1。诱导培养4周后,测定心肌样细胞转化率和细胞直径、cTroponin-I免疫组化染色、Western-blot方法,测定GATA-4蛋白表达及蛋白磷酸化水平、RT-PCR方法测定β-MHC的表达、透射电镜观察心肌样细胞超微结构特征。结果Ⅳ2组心肌样细胞直径显著增大(P<001vsⅢ组),Ⅲ、Ⅳ2组心肌样细胞转化率无显著差异(P>005)。Ⅲ、Ⅳ2组cTroponin-I染色阳性细胞数显著增多,β-MHC表达显著增高(P<001vsⅠ组),GATA-4蛋白表达及磷酸化水平均亦显著增高(P<005vsgroupⅠ、Ⅱ)。ET-1浓度为30nmol/L时,GATA-4蛋白磷酸化程度最高(P<005)。超微结构显示,Ⅲ、Ⅳ组分化的心肌样细胞可见原始肌节形成,其中,Ⅳ2组更为明显。结论5-aza及5-aza联合ET-1所诱导BMSCs分化的心肌样细胞,具有心肌细胞的生物学特征;单独采用ET-1,不能诱导BMSCs向心肌样细胞分化;ET-1在诱导BMSCs分化中,可提高GATA-4蛋白磷酸化水平,发挥促进心肌样细胞成熟的生物?

AIM: To investigate biological effect of endothelin-1 (ET-1) on differentiation of cardiomyogenic cells (CGCs) induced from rabbit bone mesenchymal stem cell (BMSCs) in vitro. METHODS: The BMSCs from shaft of femur of Japan rabbits were isolated and propagated for cell culture. Cultured BMSCs were randomly divided into six groups: Group Ⅰ: the control group (uninduced group); Group Ⅱ: the ET-1 group, added ET-1 (30 nmol/L) into culture medium; Group Ⅲ: 5-aza group, added 5-aza (10 μmol/L) into culture medium; Group Ⅳ: 5-aza+ET-1 group, after inducing with 5-aza for 3 weeks, ET-1 was added into culture medium. This group was divided into three parts-Ⅳ 1、Ⅳ 2、Ⅳ 3, and separated to add 10、 30、 50 nmol/L ET-1. During the cell culture, growing and differentiation of BMSCs were observed. After inducing for 4 weeks, the differentiation rate and the diameter of cardiomyogenic cells were calculated; Western-blot was employed to analyze the expressions of GATA-4 protein and phosphorylation level. The expression of β-MHC mRNA was assessed by RT-PCR. Immunohistochemistry staining of Troponin-I and ultrastructure observation of induced cardiomyogenic cells were also completed simultaneously. RESULTS: The cell diameters of CGCs in group Ⅳ 2 were enlarged significantly (P<0.01 vs group Ⅲ), but there was no significant differences in differentiation rate between the two groups (P>0.05). In group Ⅲ and Ⅳ 2 , the positive cells of cTroponin I staining were more, the expression of β-MHC mRNA was significantly increased (P<0.01 vs group Ⅰ)and GATA-4 protein and phosphorylation levels were significantly increased (P<0.05 vs group Ⅰ, Ⅱ). Among three concentrations of ET-1 employed, 30 nmol/L ET-1 gave rise to the highest phosphorylation level of GATA-4 in CGCs (P<0.05). In group Ⅲ and Ⅳ, the microstructure of some cells were shown: the single round or oviform nucleus located in the center of cell and the cleolus of it was obviously large. The euchromatin of it distributed evenly and few heterochromatin dispersed in it. The primitive sarcomere was also visible, especially in group Ⅳ 2. CONCLUSION: The myocardial cell characteristics of CGCs were obvious induced with 5-aza combined with ET-1. ET-1 alone could not induce BMSCs to differentiate to CGCs. Endothelin-1 can increase the GATA-4 phosphorylation level of CGCs and promote the proliferation and mature of CGCs in vitro. [