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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: To differentiate BMSCs by cardiomyocyte lysate and observe whether SMCs can express BNP and β1 receptor mRNA after receptor signaling pathway. Methods: Cardiomyocytes of neonatal rats were isolated and cardiomyocyte lysate was prepared. MSCs were isolated from the long bone marrow of adult mice and cultured for 1 week in medium containing cardiomyocyte lysate. The levels of BNP and β1 receptor mRNA in MSCs were detected qualitatively by reverse transcription polymerase chain reaction (RT-PCR), and the content of cyclic adenosine monophosphate (cAMP) in MSCs was determined by radioimmunoassay. Results: The induced group of SMCs could express BNP and β1 receptor mRNA, while the control group did not express BNP and β1 receptor mRNA. After induced by 10-8,10-7,10-6,10-5mol / L isoproterenol (ISO) for 2 h, the induced SMCs could increase intracellular cAMP content and had ISO concentration dependence (P < 0.05 or P <0.01). The induced SMCs were treated with 10-6,10-5mmol / L metoprolol (MET) for 10 min, and then combined with 1 × 10-7mol / L ISO for 110 min. The intracellular cAMP level and Compared with 10-7mol / L ISO, the difference was statistically significant (P <0.01), indicating that MET could completely block the effect of ISO on intracellular cAMP. CONCLUSION: Cardiomyocytes lysate can simulate the myocardial microenvironment to induce the differentiated SMCs to express β1 receptor and BNP mRNA in vitro and have the signal transduction pathway of post-receptor activity.