内源性多肽ECDP4对子宫内膜癌肿瘤微环境中淋巴细胞分群影响的实验研究  

The effects of endogenous peptide ECDP4 on lymphocyte subpopulations in endometrial cancer micro-environment

作　　者：布沙来木·木哈买 寸云花 季春雅 王露瑶 付子毅[2] 阮红杰[1] BUSHALAIMU·Muhamai;CUN Yunhua;JI Chunya;WANG Luyao;FU Ziyi;RUAN Hongjie(Department of Gynecology,Women's Hospital of Nanjing Medical University,Nanjing Maternity and Child Health Care Hospital,Nanjing 210004,China)

机构地区：[1]南京医科大学附属妇产医院南京市妇幼保健院妇科,南京210004 [2]南京医科大学附属第一医院妇幼研究中心乳腺病研究中心,210029

出　　处：《临床肿瘤学杂志》2023年第7期577-584,共8页Chinese Clinical Oncology

基　　金：国家自然科学基金资助项目(81972435,82173327);江苏省研究生培养创新工程研究生科研与实践创新计划资助项目(SJCX21_0646)。

摘　　要：目的探讨内源性多肽ECDP4在人外周血单核细胞(PBMCs)和子宫内膜癌Ishikawa细胞共培养体系下对PBMCs中T淋巴细胞亚群比例的影响。方法收集2020年9月至2020年11月手术切除的3例子宫内膜癌和3例配对正常子宫内膜组织标本。使用在线数据库UniProt、ProteomicsDB、MaxQB、UniProt和The Human Protein Atlas分析多肽ECDP4的前体蛋白及其生物学功能。建立人子宫内膜癌Ishikawa细胞株和新鲜人PBMCs共培养模型,分为对照组(含5%胎牛血清培养基)和实验组(含5%胎牛血清培养基中加100 ng/L内源性多肽ECDP4),共培养96 h,收集PBMCs。用流式细胞术分析PBMCs中淋巴细胞亚群比例变化。结果在正常子宫内膜组织中,ECDP4表达量为7.33×10-4±1.13×10-4,而在子宫内膜癌组织中表达量为7.30×10^(-5)±8.47×10^(-6)(P=0.0016)。生物信息学分析显示,NOVA2是ECDP4的前体蛋白,但其并不是子宫内膜癌的预后因素。流式细胞术检测显示,相较于单纯培养PBMCs,共培养体系中CD4^(+)T淋巴细胞比例下调(51.31±11.05)%(P<0.001),CD8^(+)T淋巴细胞比例下调(52.98±5.86)%(P<0.001)。相较于对照组,在共培养体系中加入ECDP4后,CD4^(+)T淋巴细胞比例上调(15.58±2.34)%(P<0.001),Ki-67的比例上调(54.60±24.5)%(P=0.0043),干扰素γ(INF-γ)的比例上调(27.92±17.25)%(P=0.0486);CD8^(+)T淋巴细胞比例上调(20.04±6.70)%(P<0.001),Ki-67的比例上调(26.57±6.42)%(P<0.001),INF-γ比例上调(87.48±40.70)%(P=0.01)。结论多肽ECDP4作为NOVA2的降解产物在子宫内膜癌中显著低表达,其能够增加PBMCs中CD4^(+)T淋巴细胞及CD8^(+)T淋巴细胞的比例,促进CD4^(+)及CD8^(+)T淋巴细胞的增殖作用,同时增加INF-γ的分泌量。内源性多肽ECDP4具有进一步应用于临床研究的价值。Objective To explore the effect of endogenous peptide ECDP4 on the T lymphocyte subpopulations in human peripheral blood mononuclear cells(PBMCs)in the co-culture system of PBMCs and endometrial cancer cell line Ishikawa.Methods Three cases of endometrial cancer and three paired normal endometrial tissue specimens were collected from surgical resection from September 2020 to November 2020.The precursor proteins of peptide ECDP4 and their biological functions was analyzed using online databases UniProt,ProteomicsDB,MaxQB,UniProt and The Human Protein Atlas.Human endometrial cancer cell line Ishikawa was co-cultured with fresh human PBMCs.The dosage of 100 ng/L endogenous peptide ECDP4 was added in the medium of the experiment,and the control group(NC)was applied with the same amount of medium containing 5%fetal bovine serum.PBMCs and supernatants were harvested after 96 h of co-culture with Ishikawa cell line.We used flow cytometry to characterize PBMCs subpopulations and their secretion of cytokine.Results In normal endometrial tissues,the expression of ECDP4 was 7.33×10^(-4)±1.13×10^(-4),while in endometrial cancer tissues the expression was 7.30×10^(-5)±8.47×10^(-6)(P=0.0016).Bioinformatics analysis showed that NOVA2 was a precursor protein for ECDP4,but it was not a prognostic factor for endometrial cancer.According to the results of flow cytometry,the proportion of CD4^(+)T lymphocytes was reduced by(51.31±11.05)%(P<0.001),and the percentage of CD8^(+)T lymphocytes was decreased by(52.98±5.86)%(P<0.001)in co-culture system compared with PBMCs alone.After adding ECDP4 into the co-culture system,the proportion of CD4^(+)T lymphocytes increased by(15.58±2.34)%(P<0.001),the secretion of Ki-67 increased by(54.60±24.5)%(P=0.0043),INF-γincreased by(27.92±17.25)%(P=0.0486);CD8^(+)T lymphocytes increased by(20.04±6.70)%(P<0.001),the secretion of Ki-67 increased by(26.57±6.42)%(P<0.001),INF-γincreased by(87.48±40.70)%(P=0.01).Conclusion Peptide ECDP4,as a degradation product of NOVA2,is significantly le

关 键 词：子宫内膜癌 内源性多肽ECDP4 外周血单个核细胞 共培养 

分 类 号：R737.33[医药卫生—肿瘤]