摘要
目的:研究转化生长因子β1(TGF-β1)诱导心肌成纤维细胞(CFs)向肌成纤维细胞(MFB)转分化过程中Notch1的变化情况,探讨在此转分化过程中Notch1的可能作用.方法:体外分离培养CFs,以10μg/LTGF-β1诱导CFs向MFB转分化.实验分为对照组,TGF-β1作用24,48,72h组,采用消化法测定羟脯氨酸(HYP)含量;免疫荧光、免疫印迹法测定α-平滑肌肌动蛋白(α-SMA)表达;实时定量PCR、免疫印迹法测定Notch1 mRNA及蛋白表达.利用γ分泌酶抑制剂DAPT(75μmol/L)阻断Notch1受体活化.实验分对照组,DAPT作用24,48,72h组,检测SMA及胶原的表达变化.结果:TGF-β1作用后,与对照组相比较α-SMA,HYP表达量随TGF-β1诱导时间的增加呈上升趋势,而Notch1则呈下降趋势,均以72h最显著(P<0.01);加入DAPT后,与对照组相比较α-SMA,HYP表达量随DAPT作用时间增加呈上升趋势,以72h最显著(P<0.01).结论:TGF-β1可以诱导CFs向MFB转分化,Notch1在此过程中的表达呈下降趋势,而DAPT阻断Notch1活化后可以促进CFs向MFB转分化.
AIM：To investigate the change of Notch1 in the transdifferentiation of cardiac fibroblasts（CFs）into myofibroblasts（MFB）induced by TGF-β1 and the possible effects of Notch1 during this progess.METHODS：CFs were obtained and cultured in vitro,and were stimulated with TGF-β1（10 μg/L）to promote the transdifferentiation of CFs into MFB.The CFs were divided into control group,24,48 and 72 h group based on the time length of TGF-β1 stimulation.Hydroxyp roline（HYP）content was detected by alkaline hydrolysis colorimetry,the expression level of α-smoothmuscle actin（α-SMA）protein was measured by Western Blot and immunofluorescence,and the expression levels of Notch1 mRNA and protein were detected by quantitative real-time PCR and Western Blot.Gamma-secretase inhibitor,N-[N-（3,5-difluorophenacetyl）-l-alanyl]-S-phenylglycine t-butyl ester,DAPT（75 μmol/L）was then used to block the activation of Notch1 receptors,and the CFs were divided into control group,24,48 and 72 h group based on the time length of DAPT stimulation.Then the expressionlevels of α-SMA and HYP were detected.RESULTS：As the time extension of TGF-β1 stimulation,the expression levels of HYP and α-SMA increased gradually while Notch1 declined compared with the control group,with the most significant effect in 72 h group（P〈0.01）.As the incubating time with DAPT prolonging,the expression levels of α-SMA and HYP rose compared with the control group,with the most significant effect in 72 h group（P〈0.01）.CONCLUSION：TGF-β1 can induce the transdifferentiation of CFs into MFB,in which the expression level of Notch1 decreased.DAPT can block the activation of Notch1 and promote the transdifferentiation.Thus,Notch1 may play an important role in the transdifferentiation of CFs into MFB,and it will possibly becomea new therapeutic target for blocking even reversing the cardiac fibrosis.
出处
《第四军医大学学报》
北大核心
2009年第22期2489-2492,共4页
Journal of the Fourth Military Medical University
基金
国家自然科学基金(30800470)
