不同浓度的窦房结细胞裂解液对兔骨髓间充质干细胞的诱导分化作用

Differentiation of rabbit bone marrow mesenchymal stem cells induced by different concentrations of lysed sinoatrial node cardiomyocytes

目的探索不同浓度的窦房结细胞裂解液对兔骨髓间充质干细胞的诱导分化作用。方法体外分离兔窦房结细胞并制备窦房结细胞裂解液。全骨髓贴壁法分离纯化兔骨髓间充质干细胞,分别加入含1、5、10倍浓度的窦房结细胞裂解液作为诱导培养基,普通培养基培养作对照,观察细胞形态变化,通过免疫荧光染色法检测超极化激活的环核苷酸门控的离子通道蛋白HCN4的表达,免疫细胞化学染色检测心肌特异性肌钙蛋白T(cTnT)的表达。结果经1、5、10倍浓度窦房结细胞裂解液诱导后的细胞均表达cTnT和HCN4;4组间HCN4阳性细胞率比较,差异有统计学意义(P<0.05)。随着细胞裂解液浓度的提高,所诱导成的阳性细胞数再逐步增加,但增加至5倍浓度以上,效果并不明显。结论在一定范围内,不同浓度的窦房结细胞裂解液能够体外诱导骨髓间充质干细胞分化为起搏细胞,但并不遵从剂量效应关系。

Objective To investigate the effect of different concentrations of sinoatrial node cell lysate on the differentiation of rabbit bone marrow mesenchymal stem cells(BMSCs).Methods Rabbit sinoatrial node cells were isolated in vitro,and the sinoatrial node cell lysate was prepared.BMSCs were isolated and purified by the whole bone marrow adherence method.The cells were cultured in a medium containing 1,5,10 times of concentration of sinoatrial node cell lysate.The morphological changes of the cells were observed in the standard medium.The hyperpolarized cyclic nucleotide-gated ion channel protein HCN4 was detected by immunofluorescence staining.The expression of cardiac troponin T(cTnT)was detected by immunocytochemical staining.Results The cells induced by 1,5,10 times concentration of sinoatrial node lysate all expressed cTnT and HCN4,and the positive rate of HCN4 was significantly different among the four groups(P<0.05).With the increase of cell lysate concentration,the number of positive cells increased gradually,but the effect was not noticeable when the concentration was more than five times.Conclusion In a certain range,different concentrations of SAN lysates can induce BMSCs to differentiate into pacemaker cells in vitro but do not comply with the dose-effect relationship.