人前列腺癌中己糖激酶-2基因对TH1/TH2细胞的影响

The impact of hexokinase-2 gene of human prostate cancer on T help 1/T help 2 cell

目的探讨在前列腺癌PC3细胞系中高表达的己糖激酶-2基因(HK2基因)对与PC3细胞系共培养体系里的辅助性T细胞1和辅助性T细胞2(TH1/TH2)的影响。方法通过shRNA构建慢病毒感染PC3细胞系产生HK2基因敲低的PC3细胞系的实验组细胞(sh-HK2),同时使用无义序列构建慢病毒感染PC3细胞系获得阴性对照细胞(NC),qPCR与Western blot验证敲低结果。培养前列腺癌细胞(NC和sh-HK2)48 h后分离癌细胞获得上清液,提取健康人外周血单个核细胞(PBMCs)。TH1空白组:PBMCs在TH1诱导条件下使用1640培养基培养48 h;TH1实验组(TH1 sh-HK2组):PBMCs在TH1诱导条件下使用培养过稳转株的上清液共培养48 h;TH1阴性对照组(TH1 NC组):PBMCs在TH1诱导条件下使用培养过阴性对照细胞的上清液共培养48 h。TH2空白组、TH2实验组(TH2 sh-HK2组)、TH2阴性对照组(TH2 NC组)诱导条件变更为TH2诱导条件,其他不变。48 h后分光光度仪检测各共培养体系中乳酸水平。然后提取各组中PBMCs使用植物血凝素-M(PHA-M)另外培养24 h,酶联免疫吸附测定法(ELISA)检测TH1诱导条件下各组中PBMCs的干扰素-γ(IFN-γ)和TH2诱导条件下各组PBMCs的白介素-4(IL-4)的分泌。结果qPCR与Western blot显示HK2基因敲低结果显著,在TH1诱导条件下的实验组中PBMCs分泌IFN-γ升高,在TH2诱导条件下的实验组中PBMCs分泌IL-4降低。实验组与对照组相比乳酸含量均下降。结论HK2基因在前列腺癌PC3中的高表达使肿瘤周围环境乳酸浓度升高,并使周围共培养体系中IFN-γ水平降低,IL-4水平升高,由此导致细胞免疫功能下降,而使针对肿瘤无效的体液免疫增强,促进肿瘤进展。

Objective To study the impact of high expression level of HK2 gene in human prostate cancer on T help 1/T help 2,which are co-cultured with human prostate cancer(PC3 cell line).Methods shRNA was used to construct a stable transgenic cells(sh-HK2) of PC3 cell line with shRNA-infected PC3 cell line induced by lentivirus infection, nonsense sequences was used to construct lentivirus, PC3 cell line was infected to obtain negative control cells(NC),then the result of knocking down was inspected through qPCR and Western blot.Isolated cancer cells to obtain supernatant after culturing PC3 cells(NC and sh-HK2) for 48 hours. Human peripheral blood mononuclear cells(PBMCs) were extracted from healthy persons. TH1 blank group: PBMCs were cultured in 1640 medium for 48 h under TH1 induction conditions, and TH1 experimental group(TH1 sh-HK2 group): PBMCs were co-cultured for 48 h in TH1 induction conditions using the supernatant of the cultured stable transgenic cells.(TH1 NC group): PBMCs were co-cultured for 48 h under the condition of TH1 induction using the supernatant of the cultured negative control cells. The induction conditions of the TH2 blank group, the TH2 experimental group(TH2 sh-HK2 group), and the TH2 negative control group(TH2 NC group) were changed to TH2 induction conditions, and the others were unchanged. After 48 hours, the lactate level in each co-culture system was detected by a spectrophotometer. PBMCs were then extracted and cultured with phytohemagglutinin-M(PHA-M) for another 24 h, then the secretion of IFN-γ of PBMCs in TH1 induction conditions and IL-4 of PBMCs in TH2 induction conditions were inspected through ELISA.Results qPCR and Western blot showed that HK2 gene knockdown was significant.In TH1 experimental group of TH1-inducing conditions,the secretion of IFN-γ of PBMCs raised. In TH2 experimental group of TH2-inducing conditions,the secretion of IL-4 of PBMCs decreased. The level of lactic acid in sh-HK2 group declined compared with NC group. Conclusion The high expression level of HK2 gene in PC3 cell line enhances lactic acid in tumor environment. At the same time, it decreases the secretion of IFN-γ and enhances the secretion of IL-4, which leads to weak cellular immune function,therefore humoral immunity that invalid for tumor immunity is enhanced, resulting in the progress of tumor.