过表达AT2R对LPS诱导的AML12细胞炎症反应的影响  

Effects of overexpression of AT2R on LPS-induced inflammatory responses in AML12 cells

作　　者：许昌勇 张世浩 魏伟[1] Xu Changyong;Zhang Shihao;Wei Wei(Institute of Clinical Pharmacology,Anhui Medical University,Key Laboratory of Anti-inflammatory and Immune Medicine,Ministry of Education,Anhui Collaborative Innovation Center of Anti-inflammatory and Immune Medicine,Hefei 230032)

机构地区：[1]安徽医科大学临床药理研究所,抗炎免疫药物教育部重点实验室,抗炎免疫药物安徽省协同创新中心,合肥230032

出　　处：《安徽医科大学学报》2023年第10期1712-1718,共7页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81871973)。

摘　　要：目的探究过表达血管紧张素Ⅱ2型受体(AT2R)对脂多糖(LPS)诱导的小鼠肝细胞(AML12细胞)炎症反应的影响。方法以乳鼠组织器官为样本,通过PCR扩增出含有EcoRⅠ和HindⅢ双酶切位点的AT2R目的基因片段,通过酶切、连接得到最终产物,挑选出的阳性克隆经测序鉴定;将pCMV-Flag-N-AT2R质粒转染至HEK 293T细胞,24 h后采用Western blot法检测Flag蛋白的表达;通过Western blot和激光共聚焦显微镜观察AML12细胞上的AT2R表达;对AML12细胞进行不同的处理,分为对照组、LPS处理组、pCMV-Flag-N-AT2R组和pCMV-Flag-N-AT2R+LPS组,通过CCK-8检测细胞活力;Western blot检测PCNA蛋白,观察细胞增殖;qPCR检测细胞炎性因子水平;Western blot检测细胞核中转录因子NF-кB(p65)表达水平。结果EcoRⅠ和HindⅢ双酶切鉴定以及测序结果表明pCMV-Flag-N-AT2R质粒构建成功,Western blot法的检测结果表明AT2R蛋白成功表达;激光共聚焦观察到AML12细胞上有AT2R受体,AT2R重组质粒可以在AML12上表达;与对照组比较,LPS处理的AML12细胞中细胞的活力和增殖能力减弱,而白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)水平升高,核转录因子NF-кB(p65)表达水平升高;与LPS组比较,pCMV-Flag-N-AT2R和LPS联合处理的AML12细胞中细胞的活力和增殖能力增强,而IL-6和TNF-α水平降低,核转录因子NF-кB(p65)表达水平降低。结论成功构建了AT2R过表达质粒,并在AML12细胞上成功表达,且AT2R可以抑制LPS诱导的AML12细胞的炎症反应。Objective AngiotensinⅡType 2 receptor(AT2R)is a major receptor of angiotensinⅡ,which has a protective effect on damage to various tissues and organs.In this study,an overexpression plasmid of AT2R was constructed to explore the effect of overexpression of AT2R on lipopolysaccharide(LPS)-induced inflammatory response in mouse hepatocytes(AML12 cells).Methods Mice tissues and organs were used as samples to amplify the gene of interest(AT2R)fragments containing EcoRⅠand HindⅢrestriction sites,and then the final product was obtained by digestion and ligation.The final positive clones were sequenced and identified.The pCMV-Flag-N-AT2R plasmid was transfected into HEK 293T cells,and the expression of Flag protein was detected by the Western blot method after 24 h.AT2R expression on AML12 cells was observed by Western blot and laser confocal microscopy.AML12 cells were treated differently and divided into control group,LPS treatment group,pCMV-Flag-N-AT2R group and pCMV-Flag-N-AT2R+LPS group,and cell viability was detected by CCK8.Western blot detected PCNA proteins and observed cell proliferation;Cytoinflammatory factor levels were detected by qPCR;Western blot detected the expression level of nuclear transcription factor NF-кB(p65)in cells.Results The identification and sequencing results of EcoR I and Hind III double restriction showed that pCMV-Flag-N-AT2R plasmid was successfully constructed,and the detection results of the Western blot method showed successful expression of AT2R protein.Laser confocal observed that there were AT2R receptors on AML12 cells,and AT2R recombinant plasmids could be expressed on AML12;Compared with the control group,the viability and proliferation ability of LPS-treated AML12 cells were weakened,while the levels of IL-6 and TNF-αincreased,and the expression level of nuclear transcription factor NF-кB(p65)increased.Compared with the LPS group,the viability and proliferation of cells in AML12 cells treated with pCMV-Flag-N-AT2R and LPS were enhanced,while the levels of IL-6 and

关 键 词：血管紧张素Ⅱ2型受体 白细胞介素-6 肿瘤坏死因子-α 脂多糖 AML12 质粒构建 

分 类 号：R34[医药卫生—基础医学]