sLAG3对人低分化胃癌MNK45细胞的抑制作用及其机制探讨

目的:探讨可溶性淋巴细胞活化基因3(sLAG3)对人低分化胃癌MNK45细胞的抗肿瘤作用及其相关机制。方法:将体外培养的MNK45细胞分为对照组和实验组,实验组加入不同浓度的sLAG3,对照组加入等体积RPMI-1640培养基,每个浓度设置3个时间点,共计12个组;采用四甲基偶氮唑盐(MTT)法检测细胞活性,流式细胞技术检测细胞凋亡,黏附实验、Transwell实验检测细胞黏附、侵袭和迁移能力;采用RT-PCR和Western Blot检测细胞CD44、CD133 mRNA和蛋白的表达水平。采用MNK45细胞建立人低分化胃癌裸鼠模型,将造模成功的荷瘤裸鼠分为对照组和实验组,实验组皮下注射不同浓度的sLAG3,对照组给予等体积的磷酸盐缓冲液(PBS),观察和测量瘤块体积和重量,流式检测外周血中的CD8^+CD28^+T细胞数量,ELISA法检测脾脏淋巴细胞分泌IL-12、INF-γ、IL-7及IL-2的水平。结果:与对照组相比,sLAG3呈剂量依赖性抑制MNK45细胞的增殖、黏附、侵袭和迁移能力;同时,sLAG3呈剂量依赖性下调MNK45细胞CD44和CD133 mRNA和蛋白的表达水平。与对照组相比,荷瘤裸鼠实验表明sLAG3呈剂量依赖性抑制瘤块生长,同时sLAG3显著上调外周血液中CD8^+CD28^+T细胞水平并促进淋巴细胞分泌IL-12、INF-γ、IL-7及IL-2。结论:本研究证明sLAG3通过调节人低分化胃癌细胞的表面分子和外周血液中CD8^+CD28^+T细胞数量及其介导的免疫反应抑制人低分化胃癌的发生和发展。

Inhibitory effect of sLAG3 on human poorly differentiated gastric cancer MNK45 cells and its mechanism

Objective:To investigate the antitumor effect of soluble LAG3（sLAG3) on human poorly differentiated gastric cancer MNK45 cells and its related mechanism.Methods:MNK45 cells cultured in vitro were divided into control group and sLAG3 group.Each group was set at 3 time points for a total of 12 groups.MTT assay was used to detect cell growth inhibition.Flow cytometry was used to detect apoptosis.Adhesion test and Transwell test were used to detect cell adhesion,invasion and migration.The expression of CD44 and CD133 mRNA was detected by RT-PCR.Western Blot was used to analyse the expression of CD44 and CD133 at protein level.The nude mice model of human poorly differentiated gastric cancer was established by MNK45 cells.The nude mice bearing tumor were divided into control group and experimental group.The experimental group was subcutaneously injected with different concentrations of sLAG3,and the control group was given the same volume of phosphate buffer（PBS),to observe and measure the volume and weight of the tumor mass,and the number of peripheral CD8+CD28+T cells was detected by flow cytometry.The levels of IL-12,INF-γ,IL-7 and IL-2 secreted by splenic lymphocytes were detected by ELISA.Results:sLAG3 could inhibit the proliferation,adhesion,invasion and migration of MNK45 cells compared with the control group.sLAG3 significantly down-regulated the expression of CD44 and CD133 genes in MNK45 cells in a dose-dependent manner.sLAG3 could inhibit the growth of tumor mass and up-regulated the level of peripheral CD8+CD28+T cells,as well as promoted the secretion of IL-12,INF-γ,IL-7 and IL-2 by T cells in vivo compared with the control group.Conclusion:This study demonstrated that sLAG3 inhibits the occurrence and development of human poorly differentiated gastric cancer by regulating the surface molecules of human poorly differentiated gastric cancer cells and the number of CD8+CD28+T cells in peripheral blood and and their mediated immune response.