乙肝肝硬化肝组织和外周血自然杀伤细胞的表型及功能特点
|
摘要
目的分析乙肝肝硬化患者肝脏和外周血自然杀伤(NK)细胞的表型和功能特点,进一步阐释人NK细胞在肝纤维化进展中的作用。方法选择2015年1月-2017年1月郑州市第一人民医院与解放军总医院第五医学中心收治的30例乙肝肝硬化患者为研究对象,同时以30例健康人为对照组。所有入组对象均抽取肝素抗凝外周静脉血,其中30例肝硬化患者中有10例通过肝组织穿刺获得肝组织标本,另有8例健康肝组织标本来源于器官捐献供体肝脏。采用流式细胞术分析两组人群肝组织和外周血NK细胞及CD56~(bright)、CD56~(dim)NK细胞亚群的频率、表型(包括活化分子及毒性分子等)和功能特点;通过与肝星状细胞(HSCs)细胞株LX-2共培养观察NK细胞对HSCs的直接杀伤作用。结果与对照组比较,肝硬化患者肝脏和外周血NK细胞频率明显降低(分别为U=8.5,P=0.006;U=184.0,P<0.001);活化分子CD69(U=102.0,P=0.009)、HLA-DR(U=82.5,P<0.001)、CD38(U=0.0,P=0.029)表达明显增高;外周血NK细胞毒性分子Granzyme B的表达明显降低(U=0.0,P=0.004);肝组织内NK细胞除肿瘤坏死因子相关凋亡诱导配体(TRAIL)表达明显增高外(U=4.0,P=0.026),穿孔蛋白、Granzyme A和Granzyme B的表达均呈下降趋势,以穿孔蛋白的下降最明显(U=4.0,P=0.034);肝脏NK细胞分泌干扰素(IFN)-γ的能力明显降低(U=2.0,P=0.032),外周血NK细胞分泌CD107a和IFN-γ的能力明显降低(分别为U=88.0,P=0.018;U=13.0,P<0.001)。纯化外周血NK细胞与LX-2细胞共培养,发现NK细胞诱导LX-2细胞发生早期凋亡(7AAD–AnnexinV~+)和晚期凋亡(7AAD~+AnnexinV~+)的比例均显著降低(分别为U=6.5,P=0.025;U=2.0,P=0.002)。结论乙肝肝硬化患者体内NK细胞频率降低,活化水平增高,分泌CD107a和IFN-γ水平降低,杀伤HSCs的能力减弱,提示患者NK细胞抗纤维化功能处于受损状态。
Objective To analyze the phenotypic and functional characteristics of hepatic and peripheral natural killer(NK) cells in HBV cirrhotic patients, and further confirm the key role of NK cells in the immunopathogenesis of liver fibrosis.Methods Thirty HBV cirrhotic patients were recruited for this study in the First People's Hospital of Zhengzhou and Fifth Medical Center of Chinese PLA General Hospital from January 2015 to January 2017, meanwhile thirty age-and sex-matched healthy individuals were enrolled as health controls. Liver biopsies were collected from 10 HBV cirrhotic patients, and 8 healthy liver tissue samples were obtained from the healthy donors whose livers were used for liver transplantation. The frequency, phenotypic and functional characteristics of hepatic and peripheral NK cells from the two groups were analyzed by using multicolor flow cytometry.The killing activities of NK cells against HSCs were explored using LX-2 cell model(a cell line of HSCs) in vitro. Results Compared with health controls, the percentages of hepatic and peripheral NK cells were reduced significantly in cirrhotic patients(U=8.5, P=0.006; U=184.0, P<0.001, respectively); the expression levels of activation markers CD69(U=102.0, P=0.009), HLADR(U=82.5, P<0.001) and CD38(U=0.0, P=0.029) were increased; the expressions of functional molecule granzyme B(U=0.0,P=0.004) was decreased in peripheral NK cells, and perforin and Granzymes were also decreased in hepatic NK cells except TRAIL that was up-regulated(U=4.0, P=0.026), especially the change of perforin(U=4.0, P=0.034); the functional decrease in IFN-γproduction(U=2.0, P=0.032) was observed in vitro in hepatic NK cells and the reduction of CD107 a degranulation and IFN-γproduction were both observed in hepatic and peripheral NK cells(U=88.0, P=0.018; U=13.0, P<0.001, respectively); purified NK cells from peripheral blood of HBV cirrhotic patients could induce less 7 AAD– Annexin V~+ early apoptotic LX2 cells and 7 AAD~+Annexin V~+ late apoptotic LX2 cells compared with those from health controls(U=6.5, P=0.025; U=2.0, P=0.002, respectively).Conclusions NK cells from HBV cirrhotic patients displayed a decreased frequency, activation increase, functional decrease in CD107 a degranulation and IFN-γ production, and cytolytic activities decrease against HSCs in response to various stimulators in vitro compared with those from health controls. These findings demonstrate an impaired anti-fibrotic function of NK cells' in HBV cirrhotic patients and further clarify the immunopathogenesis of liver fibrosis.
Objective To analyze the phenotypic and functional characteristics of hepatic and peripheral natural killer(NK) cells in HBV cirrhotic patients, and further confirm the key role of NK cells in the immunopathogenesis of liver fibrosis.Methods Thirty HBV cirrhotic patients were recruited for this study in the First People's Hospital of Zhengzhou and Fifth Medical Center of Chinese PLA General Hospital from January 2015 to January 2017, meanwhile thirty age-and sex-matched healthy individuals were enrolled as health controls. Liver biopsies were collected from 10 HBV cirrhotic patients, and 8 healthy liver tissue samples were obtained from the healthy donors whose livers were used for liver transplantation. The frequency, phenotypic and functional characteristics of hepatic and peripheral NK cells from the two groups were analyzed by using multicolor flow cytometry.The killing activities of NK cells against HSCs were explored using LX-2 cell model(a cell line of HSCs) in vitro. Results Compared with health controls, the percentages of hepatic and peripheral NK cells were reduced significantly in cirrhotic patients(U=8.5, P=0.006; U=184.0, P<0.001, respectively); the expression levels of activation markers CD69(U=102.0, P=0.009), HLADR(U=82.5, P<0.001) and CD38(U=0.0, P=0.029) were increased; the expressions of functional molecule granzyme B(U=0.0,P=0.004) was decreased in peripheral NK cells, and perforin and Granzymes were also decreased in hepatic NK cells except TRAIL that was up-regulated(U=4.0, P=0.026), especially the change of perforin(U=4.0, P=0.034); the functional decrease in IFN-γproduction(U=2.0, P=0.032) was observed in vitro in hepatic NK cells and the reduction of CD107 a degranulation and IFN-γproduction were both observed in hepatic and peripheral NK cells(U=88.0, P=0.018; U=13.0, P<0.001, respectively); purified NK cells from peripheral blood of HBV cirrhotic patients could induce less 7 AAD– Annexin V~+ early apoptotic LX2 cells and 7 AAD~+Annexin V~+ late apoptotic LX2 cells compared with those from health controls(U=6.5, P=0.025; U=2.0, P=0.002, respectively).Conclusions NK cells from HBV cirrhotic patients displayed a decreased frequency, activation increase, functional decrease in CD107 a degranulation and IFN-γ production, and cytolytic activities decrease against HSCs in response to various stimulators in vitro compared with those from health controls. These findings demonstrate an impaired anti-fibrotic function of NK cells' in HBV cirrhotic patients and further clarify the immunopathogenesis of liver fibrosis.
引文
[1] Tsochatzis EA, Bosch J, Burroughs AK. Liver cirrhosis[J].Lancet, 2014, 383(9930):1749-1761.
[2] Sun L, Gao HB, Zhao P. Research progress in metabolic activity of natural killer cells in patients with chronic hepatitis B virus infection[J]. Med J Chin PLA, 2017, 42(7):656-660.[孙丽,高会斌,赵平.慢性乙型肝炎患者自然杀伤细胞代谢活性研究进展[J].解放军医学杂志,2017, 42(7):656-660.]
[3] Wang JN, Feng H, Wang Q, et al. Killer effect of natural killer cells combined with tamoxifen on breast cancer cells in vitro and its mechanism[J]. J Jilin Univ(Med Ed), 2017, 43(2):281-287.[王迦南,冯辉,王倩,等.自然杀伤细胞联合他莫昔芬对乳腺癌细胞的杀伤作用及其机制[J].吉林大学学报(医学版),2017, 43(2):281-287.]
[4] Tosello-Trampont A, Surette FA, Ewald SE, et al. Immunoregulatory role of NK cells in tissue inflammation and regeneration[J].Front Immunol, 2017, 8:301.
[5] Wang FS, Fan JG, Zhang Z, et al. The global burden of liver disease:the major impact of China[J]. Hepatology, 2014, 60(6):2099-2108.
[6] Zhang Y, Zhang H, Elizabeth A, et al. Epidemiology of hepatitis B and associated liver diseases in china[J]. Chin Med Sci J, 2013,27(4):243-248.
[7] Shang QH, Yu JG, Xu CZ, et al. Occult hepatitis B virus infection in chronic viral hepatitis patients with non-A to E hepatitis virus infection[J]. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2008, 22(6):440-442.
[8] Lee YA, Wallace M, Friedman SL. Pathobiology of liver fibrosis:a translational success story[J]. Gut, 2015, 64(5):830-841.
[9] Ishiyama K, Ohdan H, Ohira M, et al. Difference in cytotoxicity against hepatocellular carcinoma between liver and periphery natural killer cells in humans[J]. Hepatology, 2006, 43(2):362-372.
[10] Hydes T, Abuhilal M, Armstrong T, et al. Natural killer cell maturation markers in the human liver and expansion of an NKG2C+KIR+population[J]. Lancet, 2015, 385(Suppl 1):S45.
[11] Tang L, Peng H, Zhou J, et al. Differential phenotypic and functional properties of liver-resident NK cells and mucosal ILCls[J].J Autoimmun, 2016, 67:29-35.
[12] Zheng M, Sun H, Tian Z. Natural killer cells in liver diseases[J].Front Med, 2018, 12(3):269-279.
[13] Radaeva S, Sun R, Jaruga B, et al. Natural killer cells ameliorate liver fibrosis by killing activated stellate cells in NKG2D-dependent and tumor necrosis factor-related apoptosis-inducing ligand-dependent manners[J]. Gastroenterology, 2006,130(2):435-452.
[14] Fasbender F, Widera A, Hengstler JG, et al. Natural killer cells and liver fibrosis[J]. Front Immunol, 2016, 7:19.
[15] Mantovani S, Mele D, Oliviero B, et al. NKp30 isoforms in patients with chronic hepatitis C virus infection[J].Immunology, 2015, 146(2):234-242.
[16] Glassner A, Eisenhardt M, Kramer B, et al. NK cells from HCVinfected patients effectively induce apoptosis of activated primary human hepatic stellate cells in a TRAIL-, FasL-and NKG2Ddependent manner[J]. Lab Invest, 2012, 92(7):967-977.
[2] Sun L, Gao HB, Zhao P. Research progress in metabolic activity of natural killer cells in patients with chronic hepatitis B virus infection[J]. Med J Chin PLA, 2017, 42(7):656-660.[孙丽,高会斌,赵平.慢性乙型肝炎患者自然杀伤细胞代谢活性研究进展[J].解放军医学杂志,2017, 42(7):656-660.]
[3] Wang JN, Feng H, Wang Q, et al. Killer effect of natural killer cells combined with tamoxifen on breast cancer cells in vitro and its mechanism[J]. J Jilin Univ(Med Ed), 2017, 43(2):281-287.[王迦南,冯辉,王倩,等.自然杀伤细胞联合他莫昔芬对乳腺癌细胞的杀伤作用及其机制[J].吉林大学学报(医学版),2017, 43(2):281-287.]
[4] Tosello-Trampont A, Surette FA, Ewald SE, et al. Immunoregulatory role of NK cells in tissue inflammation and regeneration[J].Front Immunol, 2017, 8:301.
[5] Wang FS, Fan JG, Zhang Z, et al. The global burden of liver disease:the major impact of China[J]. Hepatology, 2014, 60(6):2099-2108.
[6] Zhang Y, Zhang H, Elizabeth A, et al. Epidemiology of hepatitis B and associated liver diseases in china[J]. Chin Med Sci J, 2013,27(4):243-248.
[7] Shang QH, Yu JG, Xu CZ, et al. Occult hepatitis B virus infection in chronic viral hepatitis patients with non-A to E hepatitis virus infection[J]. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2008, 22(6):440-442.
[8] Lee YA, Wallace M, Friedman SL. Pathobiology of liver fibrosis:a translational success story[J]. Gut, 2015, 64(5):830-841.
[9] Ishiyama K, Ohdan H, Ohira M, et al. Difference in cytotoxicity against hepatocellular carcinoma between liver and periphery natural killer cells in humans[J]. Hepatology, 2006, 43(2):362-372.
[10] Hydes T, Abuhilal M, Armstrong T, et al. Natural killer cell maturation markers in the human liver and expansion of an NKG2C+KIR+population[J]. Lancet, 2015, 385(Suppl 1):S45.
[11] Tang L, Peng H, Zhou J, et al. Differential phenotypic and functional properties of liver-resident NK cells and mucosal ILCls[J].J Autoimmun, 2016, 67:29-35.
[12] Zheng M, Sun H, Tian Z. Natural killer cells in liver diseases[J].Front Med, 2018, 12(3):269-279.
[13] Radaeva S, Sun R, Jaruga B, et al. Natural killer cells ameliorate liver fibrosis by killing activated stellate cells in NKG2D-dependent and tumor necrosis factor-related apoptosis-inducing ligand-dependent manners[J]. Gastroenterology, 2006,130(2):435-452.
[14] Fasbender F, Widera A, Hengstler JG, et al. Natural killer cells and liver fibrosis[J]. Front Immunol, 2016, 7:19.
[15] Mantovani S, Mele D, Oliviero B, et al. NKp30 isoforms in patients with chronic hepatitis C virus infection[J].Immunology, 2015, 146(2):234-242.
[16] Glassner A, Eisenhardt M, Kramer B, et al. NK cells from HCVinfected patients effectively induce apoptosis of activated primary human hepatic stellate cells in a TRAIL-, FasL-and NKG2Ddependent manner[J]. Lab Invest, 2012, 92(7):967-977.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |