rL-RVG通过拮抗α7烟碱型乙酰胆碱受体影响胃癌细胞凋亡及增殖

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摘要目的: 探讨表达狂犬病毒糖蛋白（RVG）的重组LaSota株新城疫病毒（recombinant LaSota strain NDV expressing the rabies virus glycoprotein，rLRVG）是否通过α7烟碱型乙酰胆碱受体（α7 nicotinic acetylcholine receptor，α7nAChR）影响胃癌细胞HGC的增殖与凋亡。方法: 将rLRVG、新城疫病毒（newcastle disease virus，NDV）和PBS分别感染胃癌HGC细胞24 h后，采用蛋白质印迹法检测RVG、NDV、procaspase 3蛋白和α7nAChR在HGC细胞中的表达，免疫荧光检测α7nAChR在HGC细胞中的表达，MTT检测HGC细胞的增殖情况。将HGC细胞随机分为rLRVG组、rLRVG+α7nAChR抑制剂组、rLRVG+α7nAChR 激动剂组，NDV组、NDV+α7nAChR抑制剂组、NDV+α7nAChR激动剂组，PBS组、PBS+α7nAChR抑制剂组、PBS+α7nAChR 激动剂组；倒置显微镜下观察各组细胞形态，蛋白质印迹法检测凋亡相关蛋白procaspase 3、bax、bcl2表达水平。结果：蛋白质印迹结果显示，病毒感染HGC细胞24 h后，NDV、procaspase 3蛋白和α7nAChR在rLRVG组、NDV组的表达均显著高于PBS组（P均<0.01），RVG蛋白在rLRVG组表达显著高于NDV组和PBS组（P均<0.01）；免疫荧光显示α7nAChR在rLRVG组中明显高于NDV组和PBS组。MTT结果显示两种病毒浓度大于10-5mmol/L 对细胞的增殖有明显抑制作用(P<0.05)。倒置显微镜下可见NDV+α7nAChR抑制剂组、PBS+α7nAChR抑制剂组HGC细胞分别较NDV组、PBS组皱缩、凋亡更明显，但rLRVG组与其α7nAChR抑制剂组相比细胞皱缩、凋亡差异不明显；rLRVG+α7nAChR激动剂组、NDV+α7nAChR激动剂组、PBS+α7nAChR激动剂组HGC细胞皱缩、凋亡分别较rLRVG组、NDV组、PBS组明显减弱。蛋白质印迹显示bcl2、bax和procaspase 3蛋白在rLRVG+α7nAChR抑制剂组的相对表达量与rLRVG组无显著性差异（P均>0.05），但在NDV+α7nAChR抑制剂组、PBS+α7nAChR抑制剂组的相对表达量分别较NDV组、PBS组显著上调(P均<0.01)；rLRVG+α7nAChR 激动剂组、NDV+α7nAChR激动剂组、PBS+α7nAChR 激动剂组的bax、procaspase 3蛋白相对表达量分别较rLRVG组、NDV组和PBS组明显下调（P均<0.01），bcl2蛋白相对表达量则明显上调（P均<0.01）。结论： rLRVG感染HGC后稳定表达，可能通过拮抗α7nAChR途径促进胃癌细胞的凋亡和抑制其增殖。

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关键词 ：重组LaSota株新城疫病毒, α7烟碱型乙酰胆碱, 凋亡, 增殖

Abstract：Objective: To investigate whether rabies virus glycoprotein recombinated newcastle disease virus(rLRVG) affects gastric cancer cell apoptosis and proliferation by antagonizing α7 nicotinic acetylcholine receptor(α7nAChR). Methods: The obtained HGC cells were divided into three groups at random, the control group infected with PBS,the NDV(newcastle diseases virus) group infected with NDV and the rLRVG group infected with rLRVG for 24 hour. And the optimum concentration of NDV,rLRVG were selected by MTT.The levels of NDV, RVG，procaspase 3 proteins and α7nAChR were analysed with Western blotting and immunofluorescence. The another population of the HGC cellls was divided into nine groups randomly which were respectively infected with PBS+inhibitor,PBS,PBS+α7nAChR agonist,NDV+inhibitor, NDV,NDV+α7nAChR agonist, rLRVG+inhibitor,rLRVG,rLRVG+α7nAChR agonist. The expression of bcl2,bax and procaspase 3 were detected by Western blotting. Cell morphological changes were observed by inverted microscope. Results: RVG proteins only expressed in the rLRVG group(P<0.01), NDV, procaspase 3 proteins expressed in both the rLRVG group and NDV group.Compared with control group and NDV group,α7 nAChR protein in rLRVG group was increased(P<0.05) indicated by the Western blotting and immunofluorescence. The results of MTT assay showed that two kinds of virus concentration over 10-5mmol/L exaggerated cell proliferation inhibition. PBS+α7nAChR inhibitor group, NDV+α7nAChR inhibitor group showed that cell shrinkage and apoptosis were more obvious than those of PBS group and NDV group under inverted microscope,but there was no significant difference between the rLRVG group and rLRVG+α7nAChR inhibitor group; at the same time ,when the groups were infected by the α7nAChR agonist, the cell shrinkage and apoptosis became weaken. Then,the expression of bcl2, bax and procaspase 3 levels were increased in NDV+α7nAChR inhibitor group and PBS+α7nAChR inhibitor compared with NDV group and PBS group(P<0.01), though the diffierence of bcl2,bax and procaspase 3 between the rLRVG and rLRVG+α7nAChR inhibitor was not so obvious(P>0.05). When the groups were infected by the α7nAChR agonist, the expression of bax and procaspase3 levels were obviously decreased (P<0.01), the expression of bcl2 levels were obviously increased(P<0.01). Conclusion: rLRVG can induce the apoptosis and inhibit proliferation in gastric cancer cells by antagonizing α7nAChR.

收稿日期: 2017-10-19

通讯作者: 步雪峰（通讯作者），主任医师，硕士生导师，E-mail:xuefengbu05@163.com

引用本文:

尹超云1，2，严玉兰3，章安伟1,2，张烜烽1,2，步雪峰2. rL-RVG通过拮抗α7烟碱型乙酰胆碱受体影响胃癌细胞凋亡及增殖[J]. 江苏大学学报:医学版, 2018, 28(01): 28-33. YIN Chao-yun1,2, YAN Yu-lan3, ZHANG An-wei1,2, ZHANG Xuan-feng1,2, BU Xue-feng2.
rL-RVG regulates gastric cancer cell apoptosis and proliferation by antagonize α7 nicotinic acetylcholine receptor. Journal of Jiangsu University(Medicine Edition), 2018, 28(01): 28-33.

链接本文:

http://zzs.ujs.edu.cn/xbyxb/CN/ 或 http://zzs.ujs.edu.cn/xbyxb/CN/Y2018/V28/I01/28

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