摘要
目的:探讨瞬时受体电位通道M8(TRPM8)对胶质瘤细胞免疫逃逸的影响,并初步探究可能的作用机制。方法:通过实时荧光定量PCR法检测人正常胶质细胞株(SVGp12)与3种胶质瘤细胞株(U251、U373、T98G)中TRPM8基因表达;将U251细胞随机分为空白对照组、TRPM8-siRNA组、NC-siRNA组、pcDNA3.1-TRPM8组和pcDNA3.1-NC组,利用脂质体分别将TRPM8-siRNA、NC-siRNA、pcDNA3.1-TRPM8和pcDNA3.1-NC转染至U251细胞中,采用实时荧光定量PCR法和Western blot检测细胞中TRPM8基因和蛋白表达,CCK-8法、集落形成实验和流式细胞术检测细胞增殖、克隆形成及凋亡情况,乳酸脱氢酶(LDH)释放法检测NK92细胞对U251细胞的杀伤作用,ELISA法检测TNF-α、IL-2、IL-6、IFN-γ水平,流式细胞术、Western blot检测细胞调节区域因子1(NCR1)与自然杀伤因子蛋白46(NKP46)的表达。结果:3种胶质瘤细胞株U251、U373、T98G中TRPM8 mRNA表达水平均显著高于正常胶质细胞株SVGp12,其中U251细胞最高;与空白对照组相比,TRPM8-siRNA组TRPM8 mRNA和蛋白的相对表达量均下调(P<0.01),细胞活性下降(P<0.05),细胞克隆形成数目减少(P<0.01),细胞凋亡率升高(P<0.01),NK92细胞对U251细胞的杀伤率提高(P<0.01),细胞上清液中TNF-α、IL-2、IL-6及IFN-γ水平均下降(P<0.01),同时,NCR1、NKP46表达也明显增加(P<0.01);与空白对照组相比,pcDNA3.1-TRPM8组TRPM8 mRNA和蛋白的相对表达量均上调(P<0.01),细胞活性升高(P<0.05),细胞克隆形成数目增加而凋亡率下降(P<0.01),NK92细胞对U251细胞的杀伤率下降(P<0.01),细胞上清液中TNF-α、IL-2、IL-6及IFN-γ水平均升高(P<0.01),NCR1、NKP46表达减少(P<0.01)。结论:抑制TRPM8表达能够调控胶质瘤微环境中免疫抑制状态,增强NK92细胞杀伤U251细胞的能力,削弱肿瘤细胞的免疫逃逸能力,这可能与调控NCR1/NKP46通路相关。
Objective:To investigate the effect of transient receptor potential melastatin 8(TRPM8)on the immune escape of glioma cells,and to explore the possible mechanism of action.Methods:Real-time fluorescent quantitative PCR was used to detect the expression of TRPM8 gene in human normal glial cell line(SVGp12)and three glioma cell lines(U251,U373,T98G).The U251 cells were randomly divided into blank group,TRPM8-siRNA group,NC-siRNA group,pcDNA3.1-TRPM8 group and pcDNA3.1-NC group.The liposome method was used to transfect TRPM8-siRNA,NC-siRNA,pcDNA3.1-TRPM8 and pcDNA3.1-NC into U251 cells,real-time fluorescent quantitative PCR and Western blot were used to detect TRPM8 gene and protein expression in cells.CCK-8 method,colony formation experiment and flow cytometry were used to detect cell proliferation,clone formation and apoptosis,lactate dehydrogenase(LDH)release method was used to detect the killing of U251 cells by NK92 cells,ELISA method was used to detect TNF-α,IL-2,IL-6,IFN-γlevels,flow cytometry and Western blot were used to detect the expression of cell regulatory region factor 1(NCR1)and natural killer factor protein 46(NKP46).Results:The expression mRNA levels of TRPM8 in the three glioma cell lines U251,U373 and T98G were significantly higher than those in the normal glial cell line SVGp12,with U251 cells being the highest.Compared with blank group,the relative expressions mRNA and protein of TRPM8 in TRPM8 siRNA group were all down-regulated(P<0.01),cell activity was decreased(P<0.05),the number of cell clone formation was decreased(P<0.01),and the rate of apoptosis was increased(P<0.01),the killing rate of NK92 cells to U251 cells was increased(P<0.01),and the levels of TNF-α,IL-2,IL-6 and IFN-γin the cell supernatant were decreased(P<0.01),at the same time,the expressions of NCR1 and NKP46 were also increased significantly(P<0.01).Compared with blank group,the relative expressions mRNA and protein of TRPM8 in pcDNA3.1-TRPM8 group were up-regulated(P<0.01),cell activity was increased(P<0.05),the numbe
作者
徐华
张海萍
王虹伊
杨苗
赵钦
马蕾
XU Hua;ZHANG Haiping;WANG Hongyi;YANG Miao;ZHAO Qin;MA Lei(Xi'an International Medical Center Hospital Radiation Therapy Center,Xi'an 710100,China)
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2023年第9期1834-1840,共7页
Chinese Journal of Immunology
基金
陕西省重点研发计划项目(2020SF-928)
西安国际医学中心医院级课题项目(2021MS005)。
关键词
瞬时受体电位通道M8
胶质瘤
杀伤
免疫逃逸
肿瘤微环境
Transient receptor potential melastatin 8
Glioma
Killing
Immune escape
Tumor microenvironment
