摘要
目的前期发现多肽^(301)LARSLKT^(307)可在动物水平显著抑制胚胎期心脏发育。该研究拟进一步在P19细胞系水平探究^(301)LARSLKT^(307)抑制心肌分化的作用和机制。方法利用PepDraw Tool、Uniprot和ProtParam在线生物信息学分析网站,对多肽301LARSLKT307的结构、保守性和理化性质进行生物信息学分析。在荧光显微镜下观察FITC标记的多肽^(301)LARSLKT^(307)进入P19细胞的情况。通过倒置显微镜观察多肽^(301)LARSLKT^(307)对P19细胞分化的影响。实时定量PCR及蛋白质免疫印迹法检测多肽对心肌分化标志基因肌钙蛋白cTnT和转录因子GATA4表达的影响。实时定量PCR检测多肽对Notch1信号通路关键分子Notch1和下游Hey1及Hey2的影响。结果生物信息学分析结果显示多肽位于其前体蛋白Talin的301~307氨基酸位点,其分子量小、结构稳定、亲脂性高,且前体蛋白保守性高。荧光显微镜下可见FITC标记的多肽301LARSLKT307可以穿过细胞膜进入P19细胞内。与对照组比较,多肽组细胞部分坏死,生长形态紊乱,细胞厚薄不一。实时定量PCR与蛋白质免疫印迹法检测显示,与对照组比较,多肽组的分化相关基因GATA4及cTnT的mRNA及蛋白表达量均有不同程度下降。实时定量PCR检测显示,与对照组比较,多肽组Notch1通路的相关基因Notch1、Hey1及Hey2的mRNA表达均有不同程度下降。结论多肽^(301)LARSLKT^(307)可能是通过抑制Notch1通路关键因子的表达发挥抑制心肌分化的生物功能。
Objective Previous study has found that peptide ^(301)LARSLKT^(307) can significantly inhibit fetal cardiac development in vivo. This study intends to further explore the role and mechanism of 301LARSLKT307 in inhibiting myocardial differentiation at P19 cell. Methods The structure, conservation, physical and chemical property and precursor proteins of peptide ^(301)LARSLKT^(307) were analyzed by PepDraw Tool, Uniprot and ProtParam online. The entry of FITC-labeled peptide ^(301)LARSLKT^(307) into P19 cells was observed under a fluorescence microscope. After adding peptide 301LARSLKT307, the differentiation of P19 cells was observed by inverted microscope. The expression of troponin cTnT and transcription factor GATA4 were detected by qPCR and Western blot assay to analyze the specific marker gene of myocardium. The expressions of Notch1, Hey1 and Hey2 were detected by qPCR to analyze the effect of peptides on Notch1 pathway in P19 cell. Results Bioinformatics analysis showed that the peptide was located at the 301-307 amino acid site of its precursor protein Talin, which is a small molecule peptide with stable structure, high lipophilicity and high sequence conservation. FITC-labeled peptide ^(301)LARSLKT^(307) can enter the P19 cell under the fluorescence microscope. Compared with the control group, the cells in the peptide group were partially necrotic, with disordered growth and morphology. The qPCR and Western blot assay showed that the differentiation related genes GATA4 and cTnT in the peptide group both decreased at different levels. The qPCR showed that the Notch-related genes Notch1, Hey1 and Hey2 in the peptide group decreased at different levels. Conclusion Peptide ^(301)LARSLKT^(307) may inhibit the biological function of myocardial differentiation by inhibiting the expression of key factors of Notch1 pathway.
作者
许耿
朱金改
喻博识
韩树萍
顾海涛
余章斌
Xu Geng;Zhu Jingai;Yu Boshi(Dept of Pediatric,Women′s Hospital of Nanjing Medical University,Nanjing Maternity and Child Health Care Hospital,Nanjing 210004;Dept of Pediatric Cardiothoracic Surgery,The First Affiliated Hospital of Nanjing Medical University,Jiangsu Province Hospital,Nanjing 210029)
出处
《安徽医科大学学报》
CAS
北大核心
2021年第2期238-243,共6页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金(编号:81870240)
南京市卫生科技发展专项资金杰出青年项目(编号:JQX18010)。
