IL-12调控胃癌细胞凋亡及免疫逃逸因子分泌机制探讨

Regulation mechanism of IL-12 on apoptosis and secretion of immune escape cytokines in gastric cancer cells

目的探讨白细胞介素-12(IL-12)调控胃癌细胞凋亡及免疫逃逸因子分泌的途径。方法培养胃癌SGC7901细胞,随机分为空白对照组、感染阴性对照(NC)腺病毒的NC腺病毒组、感染IL-12腺病毒的IL-12腺病毒组、转染NC质粒的NC质粒组、转染STAT4质粒的STAT4质粒组、转染NC siRNA的si-NC组、转染STAT4siRNA的si-STAT4组、感染IL-12腺病毒并转染STAT4siRNA的IL-12腺病毒+si-STAT4组。MTS法检测细胞活力,流式细胞术检测细胞凋亡,蛋白质印迹法检测细胞中IL-12、STAT4蛋白表达,Elisa检测培养基中IL-12、肿瘤坏死因子-α(TNF-α)和干扰素-γ(IFN-γ)的分泌量;采用方差分析进行组间的统计学处理。结果IL-12腺病毒组细胞活力(0.41±0.08)较空白对照组(0.91±0.16)及NC腺病毒(0.86±0.13)降低,差异有统计学意义,F=25.123,P<0.001;凋亡率(10.95±2.85)%较空白对照组(1.86±0.32)%及NC腺病毒(2.24±0.41)%增加,差异有统计学意义,F=47.253,P<0.001。IL-12腺病毒组p-STAT4的蛋白表达量(3.23±0.62)较空白对照组(1.00±0.18)及NC腺病毒(1.05±0.22)增加,差异有统计学意义,F=54.565,P<0.001。培养基中IFN-γ、TNF-α分泌量分别为(3.09±0.74)、(3.88±0.62)ng/mg较空白对照组的(1.37±0.24)、(0.81±0.16)ng/mg及NC腺病毒组的(1.50±0.21)、(0.88±0.14)ng/mg增加,差异有统计学意义,F值分别为61.620、39.153,均P<0.001。IL-12腺病毒+si-STAT4组的细胞活力(0.76±0.12)较IL-12腺病毒组(0.40±0.08)增加,差异有统计学意义,t=5.582,P=0.001。IL-12腺病毒+si-STAT4组的细胞凋亡率(5.71±0.93)%较IL-12腺病毒组(10.95±2.85)%降低,差异有统计学意义,t=3.908,P=0.004。IL-12腺病毒+si-STAT4组培养基中IFN-γ的分泌量(2.03±0.52)ng/mg较IL-12腺病毒组(3.88±0.62)ng/mg降低,差异有统计学意义,t=5.112,P=0001;TNF-α的分泌量(1.42±0.29)ng/mg较IL-12腺病毒组(2.39±0.51)ng/mg降低,差异有统计学意义,t=3.697,P=0.006。结论IL-12通过激活STAT4途径调控胃癌细胞凋亡及免疫逃逸因子分泌。

Objective To investigate the pathway of IL-12 in regulating apoptosis and secretion of immune escape factor of gastric cancer cells.Methods SGC7901 cells were cultured and randomly divided into blank control group,NC adenovirus group infected with NC adenovirus,IL-12 adenovirus group infected with IL-12 adenovirus,NC plasmid group transfected with NC plasmid,STAT4 plasmid group transfected with STAT4 plasmid,si-NC group transfected with NC siRNA,siSTAT4 group transfected with STAT4 siRNA,IL-12 adenovirus+si-STAT4 group infected with IL-12 adenovirus and transfected with STAT4 siRNA.MTS assay was used to detect cell viability,flow cytometry was used to detect cell apoptosis,Western blot was used to detect the expression of IL-12 and STAT4 in cells,and Elisa was used to detect the secretion of IL-12,TNF-αand IFN-γin culture medium.Analysis of variance was used for statistical treatment between groups.Results Cell viability of IL-12 adenovirus group(0.41±0.08)was decreased compared with blank control group(0.91±0.16)and NC adenovirus group(0.86±0.13),the difference was statistically significant(F=25.123,P<0.001);apoptosis rate of IL-12 adenovirus group(10.95±2.85)%was increased compared with blank control group(1.86±0.32)%and NC adenovirus group(2.24±0.41)%,the difference was statistically significant(F=47.253,P<0.001);protein expression of p-STAT4 of IL-12 adenovirus group(3.23±0.62)was increased compared with blank control group(1.00±0.18)and NC adenovirus group(1.05±0.22),the difference was statistically significant,F=54.565,P<0.001;the secretion of IFN-γand TNF-αof IL-12 adenovirus group(3.09±0.74)ng/mg,(3.88±0.62)ng/mg was increased compared with blank control group(1.37±0.24)ng/mg,(0.81±0.16)ng/mg and NC adenovirus group(1.50±0.21)ng/mg,(0.88±0.14)ng/mg,the difference was statistically significant,F=61.620,P<0.001,F=39.153,P<0.001;cell viability of IL-12 adenovirus+si-STAT4 group(0.76±0.12)was increased compared with IL-12 adenovirus group(0.40±0.08),the difference was statistically significant,t=5.582,P<0.001;apoptosis rate of IL-12 adenovirus+si-STAT4 group(5.71±0.93)%was decreased compared with IL-12 adenovirus group(10.95±2.85)%,the difference wa statistically significant,t=3.908,P<0.001;the secretion of IFN-γand TNF-αof IL-12 adenovirus+si-STAT4 group(2.03±0.52)ng/mg,(1.42±0.29)ng/mg was decreased compared with IL-12 adenovirus group(3.88±0.62)ng/mg,(2.39±0.51)ng/mg,the difference was statistically significant(t=5.112,P=0001,t=3.697,P=0.006).Conclusion IL-12 regulates apoptosis and secretion of immune escape cytokines in gastric cancer cells by activating STAT4 pathway.