胃癌患者外周血及肿瘤组织中Treg细胞及亚群的研究

发布时间：2018-04-08 23:20

本文选题：胃肿瘤　切入点：调节性T细胞　出处：《西南医科大学》2017年硕士论文

【摘要】：目的:胃癌(Gastric cancer,GC)是世界上肿瘤相关性死亡的主要原因之一,而调节性T细胞(Treg cells)被认为是抑制抗肿瘤免疫促进肿瘤免疫耐受的主要因素之一。以往有关Treg细胞与胃癌的研究结果存在争议,现已发现Treg细胞是一类具有异质性的T细胞亚群,存在表型和功能的不同,这可能是导致之前结果不一致的原因。而目前鲜有关于这类Treg亚群细胞与胃癌的研究报道,因此本次研究通过检测胃癌患者外周血以及肿瘤组织中Treg细胞及亚群的表达,探索Treg细胞及亚群与胃癌患者各临床参数的相关性。方法:搜集胃癌患者和健康对照组外周血、同一患者的胃癌组织和癌旁正常组织,制备、分离、提纯单个核细胞,标记表面抗体CD3、CD4、CD45RA、CD25和核内抗体Foxp3。再根据CD45RA和Foxp3的表达将Treg细胞分为CD45RA+Foxp3lo(rTreg),CD45RA-Foxp3hi(aTreg)和CD45RA-Foxp3lo(non-Treg)三种亚群。流式细胞仪检测分析各个组Treg及亚群细胞占CD4+细胞的比例。Graph Pad Prism统计软件(5.0版本)统计分析数据,Kruskal-wallis检验(多组)、Mann-Whitney U检验(两组)比较各组间的差异是否具有统计学意义。结果:胃癌患者外周血(GC-PBMC)及肿瘤浸润淋巴细胞(TIL)中总的CD4+CD25+Foxp3+Treg细胞占CD4+T细胞比例要比健康对照组(HD-PBMC)和癌旁正常组织浸润淋巴细胞(NIL)的明显增加(P0.0001,P0.0001),其差异有统计学意义。在外周血中胃癌患者的aTreg和non-Treg细胞比例较健康对照组明显增加(P0.0001,P0.0001),差异有统计学意义。在组织浸润淋巴细胞中,TIL-aTreg和TIL-non-Treg细胞比例较NIL明显增加(P0.0001,P0.001)。分析Treg细胞及其亚群与各临床病理参数关系发现,仅发现肿瘤转移(淋巴转移和远处转移)与Treg及亚群细胞相关,而与肿瘤位置、大小、分化程度、分期以及血清CEA浓度无明显相关性。出现转移者无论在PBMC还是TIL中总的CD4+CD25+Foxp3+Treg细胞所占比例均较无转移者明显增加(P0.05,P0.01),亚群细胞中只有转移患者TIL中的aTreg细胞所占比例较无转移者明显增加(P0.01)。结论:我们的研究证实了在胃癌患者外周血和TIL中Treg细胞所占比例明显增加,以aTreg细胞增加最为明显。而且只有TIL中增加的aTreg细胞与肿瘤转移有关,提示在肿瘤微环境中起主要抑制抗肿瘤免疫的是TIL中的aTreg细胞,促使疾病的进展。所以在今后的免疫靶向治疗中,局部地选择性消除这类亚群细胞而非消除其他亚群细胞和(或)全身性的消除整个Treg细胞将有望成为一项有效治疗胃癌的手段。同时我们还发现胃癌患者外周血及TIL中的non-Treg细胞也是增加的,但与疾病无明显关联,所以在以后的研究中,我们需要鉴别不同的Treg亚型细胞而非整个Treg细胞与肿瘤的关系,这样才有可能得出更为准确的结果。
[Abstract]:Objective: Gastric cancer is one of the major causes of tumor-related death in the world, and regulatory T cell line Treg cells is considered to be one of the main factors to inhibit anti-tumor immunity and promote tumor immune tolerance.Previous studies on Treg cells and gastric cancer have been controversial. It has been found that Treg cells are a class of heterogeneous T cell subsets with different phenotypes and functions, which may be the cause of previous inconsistent results.However, there are few reports on the expression of Treg cells and subsets in peripheral blood and tumor tissues of patients with gastric cancer.To explore the correlation between Treg cells and subsets and clinical parameters in patients with gastric cancer.Methods: the peripheral blood samples of patients with gastric cancer and healthy controls were collected, and the gastric cancer tissues and adjacent normal tissues of the same patients were collected. Mononuclear cells were prepared, isolated, purified, and labeled with surface antibody CD3p4, CD45RACD25 and intranuclear antibody Foxp3.According to the expression of CD45RA and Foxp3, Treg cells were divided into three subgroups: CD45RA Foxp3losrTrega (CD45RA-Foxp3hia Trega) and CD45RA-Foxp3losl (non-Tregt).Flow cytometry was used to analyze the ratio of Treg and subgroup cells to CD4 cells. The statistical analysis data were Kruskal-wallis test (Mann-Whitney U test).Results: the percentage of total CD4 CD25 Foxp3 Treg cells in peripheral blood of gastric cancer patients was significantly higher than that in healthy controls (HD-PBMC) and adjacent normal tissues (P 0.0001 / P 0.0001).The percentage of aTreg and non-Treg cells in peripheral blood of gastric cancer patients was significantly higher than that of healthy controls.The proportion of TIL-a Treg and TIL-non-Treg cells in infiltrating lymphocytes was significantly higher than that in NIL.By analyzing the relationship between Treg cells and their subsets and clinicopathological parameters, it was found that only tumor metastasis (lymphatic metastasis and distant metastasis) was associated with Treg and subgroup cells, but with tumor location, size and differentiation degree.There was no significant correlation between stages and serum CEA levels.The percentage of total CD4 CD25 Foxp3 Treg cells in both PBMC and TIL was significantly higher than that in patients without metastasis, and the percentage of aTreg cells in TIL of patients with metastasis was significantly higher than that of patients without metastasis.Conclusion: our study confirmed that the proportion of Treg cells in peripheral blood and TIL was significantly increased in gastric cancer patients, especially in aTreg cells.Moreover, only the increase of aTreg cells in TIL is related to tumor metastasis, suggesting that aTreg cells in TIL mainly inhibit anti-tumor immunity in tumor microenvironment, and promote the progression of the disease.Therefore, in the future, the local selective elimination of these subsets rather than the elimination of other subsets and / or the elimination of the whole Treg cell may be an effective method for the treatment of gastric cancer.We also found that the number of non-Treg cells in peripheral blood and TIL was increased in patients with gastric cancer, but there was no significant association with the disease, so in future studies, we need to distinguish the relationship between different Treg subtypes rather than the whole Treg cell.This makes it possible to arrive at more accurate results.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2

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