原发性胆汁性肝硬化高表达基因TPSAB1与CD4+T细胞增殖的相关性研究  

Correlation between the expression of TPSAB1 and the proliferation of CD4+T cells in primary biliary cirrhosis

作　　者：程亮 张葆康 曹佳伟 CHENG Liang;ZHANG Baokang;CAO Jiawei(Department of Labratory Medicine,Qingdao Chengyang District People's Hospital,Qingdao 266109,China;Department of Labratory Medicine,Qingdao Central Hospital,Qingdao 266000,China;Department of Labratory Medicine,Qingdao Chest Hospital,Qingdao 266043,China)

机构地区：[1]青岛市城阳区人民医院检验科,266109 [2]青岛市中心医院检验科,266000 [3]青岛市胸科医院检验科,266043

出　　处：《免疫学杂志》2024年第2期175-180,共6页Immunological Journal

摘　　要：目的本研究分析PBC患者与健康对照者血浆基因表达谱差异,以及差异基因类胰蛋白酶Alpha/Beta 1(tryptase Alpha/Beta 1,TPSAB1)对PBC患者外周血单个核细胞(peripheral blood mononuclear cell,PBMC)来源的幼稚CD4+T细胞增殖的影响。方法使用3名PBC患者和3名健康对照者的血浆进行微阵列分析;通过免疫细胞分离试剂盒从PBMC中分离自然杀伤细胞(natural killer,NK)细胞、B细胞、幼稚CD4+T和幼稚CD8+T细胞;通过q RT-PCR分析NK细胞、B细胞、幼稚CD4+T和幼稚CD8+T细胞中TPSAB1的表达水平;采用蛋白免疫印迹分析靶向TPSAB1基因的短发夹RNA(short hairpin RNA,sh RNA)干扰后CD4+T细胞中TPSAB1的蛋白表达水平;采用WST-1增殖试剂盒和5-乙炔基-2′-脱氧尿苷(5-Ethynyl-2’-deoxyuridine,Ed U)染色法检测幼稚CD4+T细胞在体外CD3/CD28刺激后的增殖情况;流式细胞仪测定幼稚CD4+T细胞的凋亡;通过酶联免疫吸附(enzyme linked immunosorbent assay,ELISA)实验测定在体外刺激后敲低TPSAB1对幼稚CD4+T细胞干扰素-γ(interferon-γ,IFN-γ)、白介素-22(interleukin-4,IL-22)和IL-17分泌的影响。结果PBC患者NK细胞和幼稚CD4+T细胞TPSAB1表达水平显著高于健康对照者;与对照组比较,PBC患者的幼稚CD4+T细胞在CD3/CD28刺激后,显示出更强的增殖能力。然而,2组间细胞凋亡无差异。PBC患者的IL-22、IL-17和IFN-γ分泌水平明显升高。与对照组比较,敲低TPSAB1可抑制幼稚CD4+T细胞在CD3/CD28刺激下的增殖能力,同时降低IL-22、IL-17和IFN-γ分泌水平。结论抑制TPSAB1蛋白表达水平可阻断CD4+T细胞的增殖,提示其可能成为治疗PBC的新靶点。This study performed to investigate the differences of plasma gene expression profiles between primary biliary cirrhosis(PBC)patients and healthy controls,and the effect of tryptase Alpha/Beta 1(TPSAB1)on the proliferation of naive CD4+T cells derived from peripheral blood mononuclear cell(PBMC)in patients with PBC.The plasma samples of three PBC patients and three healthy controls were analyzed by microarray;Immune cell isolation kits were used to isolate natural killer(NK)cells,B cells,naive CD4+T and naive CD8+T cells from PBMC;The expression level of TPSAB1 in NK cells,B cells,naive CD4+T cells and naive CD8+T cells were analyzed by qRT-PCR;The expression of TPSAB1 in CD4+T cells was analyzed by Western blotting after interference by short hairpin RNA(shRNA)targeting TPSAB1.WST-1 proliferation kit and 5-Ethynyl-2’-deoxyuridine(EdU)staining assay were used to detect the proliferation of naive CD4+T cells after CD3/CD28 stimulation in vitro.The apoptosis of CD4+T cells was measured by flow cytometry.Enzyme linked immunosorbent assay(ELISA)was used to determine interferon-γ(IFN-γ),interleukin-4(IL-22),and IL-17 secretion in TPSAB1 knock-down CD4+T cells.Data showed that the expression level of TPSAB1 in NK cells and naive CD4+T cells from PBC patients were significantly higher than those of healthy control group.Naive CD4+T cells from PBC patients showed enhanced proliferative capacity after CD3/CD28 stimulation compared with controls.However,there was no difference in apoptosis between the two groups.The levels of IL-22,IL-17 and IFN-γwere significantly increased in PBC patients.Compared with the control group,knockdown of TPSAB1 inhibited the proliferation ability of naive CD4+T cells under CD3/CD28 stimulation.At the same time,the levels of IL-22,IL-17 and IFN-γwere decreased.In conclusion,inhibition of TPSAB1 protein expression can block the proliferation of naive CD4+T cells,suggesting that TPSAB1 may be a new target for the treatment of PBC.

关 键 词：原发性胆汁性肝硬化 TPSAB1 CD 4+T细胞 IFN-Γ IL-22 IL-17 

分 类 号：R575.2[医药卫生—消化系统]