【作者】 陈刚； 游婷婷； 林旭； 林丽香；

【Author】 Gang Chen;Tingting You;Xu Lin;Lixiang Lin;Department of Endocrinology,Fujian Provincial Hospital;

【机构】 福建省立医院内分泌科；

【摘要】 目的:构建糖原合成酶激酶3β(GSK-3β)特异的RNAi腺病毒表达载体,从转录后水平抑制GSK-3β基因表达,为研究Wnt/GSK-3β/β-连环蛋白(β-catenin)信号通路提供技术工具;并利用构建的RNAi腺病毒感染原代人脐静脉内皮细胞(HUVEC),观察Wnt/GSK-3β/β-catenin信号通路对高游离脂肪酸诱导的血管内皮细胞增殖和凋亡的影响。方法:设计合成二对针对GSK-3βmRNA不同位点的shRNA编码序列,克隆到腺病毒穿梭质粒中,然后通过体外同源重组构建重组RNAi腺病毒表达载体;在HEK293A细胞中包装并扩增病毒、空斑实验法进行病毒滴度测定,获得高滴度的病毒上清;腺病毒感染HUVEC,Western Blot和免疫细胞化学法检测其对GSK-3β蛋白表达的抑制效应,并进一步检测对细胞内β-catenin蛋白水平的影响;用游离脂肪酸(FFA)培养细胞,RNAi腺病毒抑制GSK-3β表达,溴脱氧尿苷(BrDU)法检测细胞增殖,流式细胞仪检测细胞凋亡,探讨Wnt/GSK-3β/β-catenin信号通路对高游离脂肪酸作用下HUVEC增殖和凋亡的影响。结果:1.成功构建了针对GSK-3β基因一个位点的RNAi腺病毒表达载体,经PCR和测序鉴定干扰片断的碱基序列及插入位点完全正确;2.在HEK293A细胞中包装、扩增腺病毒,获得高滴度腺病毒上清;3.构建的RNAi腺病毒感染HUVEC后可以高效抑制GSK-3β蛋白的表达,且对GSK-3β蛋白的抑制作用可持续六天以上;4.构建的RNAi腺病毒感染HUVEC后可以明显增加细胞内β-catenin的蛋白水平,且随着MOI值增加和时间延长β-catenin蛋白量进一步增加;5.腺病毒和/或FFA(0.75 mmol/L)干预HUVEC,72h、96h后Brdu和流式细胞检测,FFA组与正常细胞相比,细胞增殖率明显下降,细胞凋亡率明显增加;RNAi腺病毒组与空病毒组相比,细胞增殖率明显增加,细胞凋亡率差别不明显;RNAi腺病毒+FFA组与FFA组相比,细胞增殖率增加,细胞凋亡率减少;空病毒+FFA组与FFA组相比,细胞增殖率和凋亡率差别均不明显。结论:构建的GSK-3β特异的RNAi腺病毒能够有效抑制GSK-3β基因的表达,增加细胞内β-catenin蛋白水平,可以作为研究Wnt/GSK-3β/β--catenin信号通路的重要技术手段;上调Wnt/GSK-3β/β--catenin信号通路可保护高游离脂肪酸诱导的血管内皮细胞损伤。更多还原

【Abstract】 Free fatty acids(FFA)-induced proliferation and apoptosis was studied in human umbilical vein endothelial cells(HUVECs).A recombinant adenovirus containing a RNAi cassette targeting the GSK-3 β gene was produced and its silencing effect on GSK-3 p gene was detected by Western blot analysis and immunohistochemistry assay in HUVECs.The effect of the RNAi on the protein level ofβ-catenin was explored by transfecting the RNAi adenovirus to inhibit the expression of GSK-3 βprotein.The subsequent effect on the Wnt/GSK-3 β/β-catenin signal pathway and on proliferation and apoptosis of HUVECs cultured with FFAs,was analyzed by BrdU assay and flow cytometery in order to explore the possible connection between the signaling pathway and FFA-induced proliferation and apoptosis.The Western blot results showed that the expression of GSK-3 P protein in HUVECs could be inhibited efficiently by the RNAi adenovirus,and that the protein level of β-catenin was increased by RNAi adenovirus transfection.The results of the BrdU assay and flow cytometer suggested that knockdown of GSK-3β with the RNAi adenovirus may stimulate the proliferation of HUVECs.Apoptosis was observed in HUVECs exposed to FFAs(0.75 mmol/L) for 72 h,and this effect could be partly reversed when interfering with the RNAi adenovirus.It may be concluded that the RNAi adenovirus specific to GSK-3 P may partly protect HUVECs from apoptosis induced by FFAs.Up-regulation of the Wnt/ P-catenin signal pathway can partly reverse FFA-induced apoptosis in HUVECs.更多还原