[1]林筝鸣 钱航 李东锋 许浩 陈继舜 闵新文 陈俊 杨汉东.胰高血糖素样肽-1受体敲除H9c2细胞株建立及其抗凋亡作用初探[J].心血管病学进展,2022,(9):852-859.[doi:10.16806/j.cnki.issn.1004-3934.2022.09.019]
　LIN Zhengming,QIAN Hang,LI Dongfeng,et al.Establishment of Glucagon-Like Peptide-1 Receptor Knockout H9c2 Cell Line and Its Anti-Apoptotic Effect[J].Advances in Cardiovascular Diseases,2022,(9):852-859.[doi:10.16806/j.cnki.issn.1004-3934.2022.09.019]
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胰高血糖素样肽-1受体敲除H9c2细胞株建立及其抗凋亡作用初探()
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《心血管病学进展》[ISSN:51-1187/R/CN:1004-3934]

卷:
期数:
2022年9期
页码:
852-859
栏目:
出版日期:
2022-09-25

文章信息/Info

Title:
Establishment of Glucagon-Like Peptide-1 Receptor Knockout H9c2 Cell Line and Its Anti-Apoptotic Effect
作者:
林筝鸣1; 2 钱航2 李东锋2 许浩2 陈继舜2 闵新文2 陈俊2 杨汉东2
(1.锦州医科大学国药东风总医院研究生培养基地,湖北 十堰 442008;2.湖北医药学院附属国药东风总医院,湖北 十堰 442008)
Author(s):
LIN Zhengming1; 2; QIAN Hang2; LI Dongfeng2; XU Hao2; CHEN Jishun2; MIN Xinwen2; CHEN Jun2; YANG Handong2
(1.Dongfeng Hospital Graduate Training Base,Jinzhou Medical University,Shiyan 442008,Hubei,China; 2. Sinopharm Dongfeng General Hospital,Hubei University of Medicine,Shiyan 442008,Hubei,China)
关键词:
CRISPR/Cas9; GLP-1R; 甲基乙二醛; H9c2心肌细胞; 线粒体; 凋亡
Keywords:
CRISPR/Cas9; GLP-1R; Methylglyoxal; H9c2; Mitochondrial;  Apoptosis
DOI:
10.16806/j.cnki.issn.1004-3934.2022.09.019
摘要:
目的 通过CRISPR/Cas9技术构建大鼠H9c2心肌细胞胰高血糖素样肽-1受体（GLP-1R）基因敲除的稳转株，探讨甲基乙二醛对其凋亡的影响。方法 利用CRISPR/Cas9技术敲除H9c2心肌细胞的GLP-1R基因建立GLP-1R-/- H9c2细胞系，单克隆后通过测序和免疫印迹进行鉴定。H9c2细胞为野生型（WT）组，GLP-1R-/- H9c2细胞为GLP-1R-/-组。采用免疫荧光对两组细胞进行染色，电子显微镜观察细胞形态；采用MGO对两组细胞进行干预，利用CCK-8检测细胞活力，JC-1检测线粒体膜电位，实时荧光定量PCR检测GLO1、Bax、Bcl-2和caspase-3 mRNA的表达。结果 基因测序及免疫印迹结果证实，GLP-1R-/- H9c2细胞系构建成功；免疫荧光染色显示GLP-1R-/-组细胞形态较WT组显著缩小（P＜0.01）；CCK-8结果显示，GLP-1R-/-组细胞较WT组显著降低（P＜0.01）；JC-1结果显示，GLP-1R-/-组细胞的线粒体膜电位损伤显著高于WT组（P＜0.01）；实时荧光定量PCR结果显示，GLP-1R-/-组细胞的Bax mRNA表达显著上调（P＜0.01），Bcl-2 mRNA表达显著下调（P＜0.01）。而caspase-3 mRNA和GLO1 mRNA的表达在两组细胞间无显著差异（P＞0.05）。结论 在GLP-1R-/-基因敲除的H9c2细胞系中，GLP-1R蛋白缺失会影响H9c2细胞的细胞形态，降低细胞活力，损伤线粒体膜电位，增强MGO诱导的凋亡，从而证明GLP-1R蛋白在H9c2细胞中的保护作用。
Abstract:
Objective To investigate the effect of methylglyoxal(MGO) on the apoptosis of stable strains of glucagon-like peptide-1 receptor(GLP-1R) gene knockout H9c2 cells which was constructed by CRISPR/Cas9 technology. Methods GLP-1R-/- H9c2 cell line was established by knocking out the GLP-1R gene of H9c2 cells by CRISPR/Cas9 technology,and identification was carried out by sequencing and western blot after monoclonalization. H9c2 cells are wild-type(WT) group and GLP-1R-/- H9c2 cells are GLP-1R-/- group. Two groups of cells were stained with immunofluorescence,and cell morphology was observed by electron microscopy. MGO was used to intervene in both groups of cells,cell viability was detected by CCK-8,mitochondrial membrane potential(MMP) was detected by JC-1,and real-time PCR was used to detect the expression of GLO1, Bax, Bcl-2, caspase-3 mRNA. Results Gene sequencing and Western blot results confirmed that the GLP-1R-/- H9c2 cell line was successfully constructed. Immunofluorescence staining showed that the cell morphology of GLP-1R-/- group was significantly smaller than that of WT group(P＜0.01). The CCK-8 results showed that the cell viability of GLP-1R-/- group was significantly reduced compared with that of WT group(P＜0.01). The JC-1 results showed that the MMP damage of GLP-1R-/- group cells was significantly higher than that of WT group(P＜0.01). The real-time PCR results showed that the Bax mRNA expression of GLP-1R-/- group cells was significantly upregulated(P＜0.01),and Bcl-2 mRNA expression was significantly downregulated(P＜0.01),while there was no significant difference in caspase-3 mRNA and GLO1 mRNA expression between the two groups(P＞0.05). Conclusion In the H9c2 cell line with GLP-1R-/- gene knockout,the deletion of GLP-1R protein affects the cell morphology of H9c2 cells,reduces cell viability,damages the MMP,enhances MGO-induced apoptosis,thereby demonstrating the protective role of GLP-1R protein in H9c2 cells.

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备注/Memo

备注/Memo:
收稿日期：2022-02-15?div>?金项目：国家自然科学基金（81573244）；湖北省卫健委科研项目（WJ2021M061）；十堰市科技局科研项目（21Y71）；湖北医药学院人才启动金资助计划项目（2018QDJZR04）
更新日期/Last Update: 2022-10-24