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

DONG Hong-yan1, ZHOU Zhong-xin2, ZHANG Zhong-ming2 (1The Center of Neurobiological Research,?2?Department of Thoracic Surgery, Affiliated Hospital, Xuzhou Medical College, Xuzhou, 221002, China)  

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 (P0.01 vs group Ⅲ), but there was no significant differences in differentiation rate between the two groups (P0.05). In group Ⅲ and Ⅳ 2 , the positive cells of cTroponin I staining were more, the expression of β-MHC mRNA was significantly increased (P0.01 vs group Ⅰ)and GATA-4 protein and phosphorylation levels were significantly increased (P0.05 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 (P0.05). 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. [

【Key Words】： Bone mesenchymal stem cells Cell culture Induction Endothelin
【Fund】： 江苏省教育厅资助课题(No.01KJB310008)
【CateGory Index】： R329.2

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