心肌细胞裂解液诱导骨髓间充质干细胞向心肌细胞的分化作用

Differentiation of mesenchymal stem cells into cardiomyogenic cells under the induction of myocardial cell lysate

目的:用心肌细胞裂解液体外诱导分化骨髓间充质干细胞(SMCs),观察SMCs能否表达脑钠肽(BNP)及β1受体mRNA,是否具有受体后信号转导通路。方法:分离新生乳鼠的心肌细胞并制成心肌细胞裂解液,自成年小鼠长骨骨髓中分离MSCs,并用含有心肌细胞裂解液的培养基培养1周。用逆转录聚合酶链反应方法定性检测MSCs中BNP及β1受体mRNA水平,用放射免疫方法测定MSCs中环磷酸腺苷(cAMP)的含量。结果:诱导组SMCs能表达BNP及β1受体mRNA,对照组不表达BNP和β1受体mRNA。诱导的SMCs经10-8,10-7,10-6,10-5mol/L异丙肾上腺素(ISO)作用2 h后,均能增加细胞内cAMP含量,且具有ISO浓度依赖性(P<0.05或P<0.01)。诱导的SMCs经不同浓度的美托洛尔(MET)10-6,10-5mmol/L作用10 min后,和1×10-7mol/L ISO再一起作用110 min后,测定细胞内cAMP含量与空白组比较,差异无统计学意义;与10-7mol/L ISO作用结果比较,差异有统计学意义(P<0.01),表明MET能完全阻断ISO升高细胞内cAMP作用。结论:心肌细胞裂解液能体外模拟心肌微环境诱导分化SMCs表达β1受体及BNP mRNA,并具有受体后活性的信号转导通路。

Objective： In order to understand the effects of cardiac microenviroment on the differentiation of bone marrow mesenchymal stem cells（MSCs） into myocardial-like cells, we stimulated the cardiac microenviroment in vitro by adding myocardial cell lysate into the culture system of MSCs and investigated whether brain na- triuretic peptide（BNP） and β1 adrenergic receptor （AR） are expressed in cultured SMCs, and if so, whether they have downstream signaling systems. Method： Myocardial ceils isolated from newly born rats were lysed by repeated freezing and defrosting. MSCs isolated from adult rat were cultured in two different systems： medium with myocardial cell lysate and control medium. RT-PCR was used to measure BNP and β1 AR mRNA expression in cultured MSCs. Cyclic adenosine monophosphate （cAMP） in myocardial cell lysate cultured MSCs was measured by radio- immunoassay. Result：There were not expression of BNP and βl AR mRNA in normal cultured MSCs. After induced by myocardial cell lysate, BNP and βl AR mRNA were expressed. Isoproterenol augmented cAMP in cultured MSCs, which was fully inhibited by metoprolol. Conclusion：The normal cultured rat MSCs didn＇t express BNP and β1 AR mRNA, after treated with myocardial cell lysate, cultured rat MSCs expressed BNP and β1 AR mRNA. β1 AR had functional signal transduction pathway.