摘要
目的:探讨T细胞免疫球蛋白黏蛋白分子3(Tim-3)对宫颈癌体内外生长进展及免疫微环境的影响。方法:免疫组化染色和qRT-PCR检测宫颈癌组织及癌旁组织中Tim-3表达;将HeLa细胞随机分对照组、NC shRNA组(转染阴性对照NC shRNA慢病毒质粒)、Tim-3 shRNA组(转染Tim-3 shRNA慢病毒质粒),qRT-PCR和Western blot检测转染效果,CCK-8检测细胞增殖活性,EdU染色检测细胞增殖,Transwell检测细胞迁移与侵袭;注射转染Tim-3 shRNA或NC shRNA慢病毒质粒的HeLa细胞构建移植瘤裸鼠模型,定时测量肿瘤体积,21 d后抽取腹主动脉血,称量瘤体质量,HE染色、TUNEL染色和免疫组化染色检测肿瘤组织生长,ELISA检测血清TNF-α、IL-1β、IL-6、IL-12表达,流式细胞术检测肿瘤浸润性免疫细胞亚群变化。结果:宫颈癌组织Tim-3表达较癌旁组织显著升高(P<0.01);转染成功获得Tim-3低表达的HeLa细胞,与对照组比较,Tim-3 shRNA组HeLa细胞增殖活性下降(P<0.01),EdU标记阳性细胞减少(P<0.01),细胞迁移数和侵袭数均减少(P<0.01);与注射转染NC shRNA慢病毒质粒的HeLa细胞组裸鼠比较,注射转染Tim-3 shRNA的HeLa细胞组裸鼠肿瘤体积减小(P<0.01),肿瘤质量降低(P<0.01),肿瘤组织内细胞凋亡数增多,增殖数减少,血清TGF-β1、IL-10含量下降(P<0.01),IL-2、IL-6含量升高(P<0.01),脾脏组织CD4^(+)T、CD8^(+)T细胞比例均升高(P<0.01)。结论:Tim-3在宫颈癌组织中高表达,降低Tim-3表达能够抑制宫颈癌体内外生长,并可能对肿瘤免疫微环境起一定调控作用。
Objective:To investigate effect of T cell immunoglobulin mucin molecule 3(Tim-3)on growth,progression and immune microenvironment of cervical cancer in vivo and in vitro.Methods:Immunohistochemical staining and qRT-PCR were used to detect expression of Tim-3 in cervical cancer tissues and adjacent tissues;HeLa cells were randomly divided into control group,NC shRNA group(transfected with negative control NC shRNA lentiviral plasmid)and Tim-3 shRNA group(transfected with Tim-3 shRNA lentiviral plasmid),qRT-PCR and Western blot were used to detect transfection effect,CCK-8 was used to detect cell proliferation activity,EdU staining was used to detect cell proliferation,Transwell was used to detect cell migration and invasion;HeLa cells transfected with Tim-3 shRNA or NC shRNA lentiviral plasmid were injected to construct a nude mouse model of xenograft tumor,tumor volume was measured regularly.Abdominal aortic blood was collected 21 days later,and tumor mass was weighed,growth of tumor tissue was detected by HE staining,TUNEL staining and immunohistochemical staining,ELISA was used to detect serum TNF-α,IL-1β,IL-6,IL-12 expressions,flow cytometry was used to detect changes in tumor infiltrating immune cell subsets.Results:Tim-3 expression in cervical cancer tissues was significantly higher than that in adjacent tissues(P<0.01);HeLa cells with low Tim-3 expression were successfully obtained after transfection.Compared with control group,proliferation activity of HeLa cells in Tim-3 shRNA group was decreased(P<0.01),EdU-labeled positive cells were decreased(P<0.01),numbers of cell migration and invasion were reduced(P<0.01);compared with nude mice in HeLa cell group transfected with NC shRNA lentiviral plasmid,tumor volume of nude mice in HeLa cell group transfected with Tim-3 shRNA was reduced(P<0.01),tumor quality was reduced(P<0.01),number of cell apoptosis was increased,number of proliferation was decreased,contents of TGF-β1 and IL-10 in serum were decreased(P<0.01),contents of IL-2 and IL-6 were increased(P<0.01),proportions of CD4^(+)T and CD8^(+)T cells in spleen tissues were increased(P<0.01).Conclusion:Tim-3 is highly expressed in cervical cancer tissues.Reducing expression of Tim-3 can inhibit growth of cervical cancer in vitro and in vivo,and may play a certain regulatory role in tumor immune microenvironment.
作者
陈秀敏
吴熊军(指导)
许文彬
邓乾葆
CHEN Xiumin;WU Xiongjun;XU Wenbin;DENG Ganbao(Department of Obstetrics and Gynecology,the Second Affiliated Hospital of Hainan Medical College,Haikou 570311,China)
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2023年第11期2282-2287,2293,共7页
Chinese Journal of Immunology
基金
海南省卫生健康行业科研项目(18A200078)。