亚砷酸钠对人淋巴细胞白细胞介素和穿孔素及颗粒酶B的影响
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摘要
目的探讨亚砷酸钠体外染毒对人淋巴细胞白细胞介素-6(interleukin-6,IL-6)、IL-10、穿孔素(perforin,PFP)和颗粒酶B(granzyme B,Gr B)水平的影响。方法采集健康成年人外周血,密度梯度离心分离出淋巴细胞,分别暴露于含终浓度为0.00(对照)、5.00、10.00、20.00、30.00μmol/L亚砷酸钠的培养基染毒12、24、48 h。采用HE染色法鉴定染毒模型;MTT比色法检测细胞存活率;ELISA法检测IL-6、IL-10、PFP和Gr B的水平。结果与不同时间的对照组相比,5.00μmol/L亚砷酸钠染毒组淋巴细胞的存活率均升高,而10.00~30.00μmol/L亚砷酸钠染毒组淋巴细胞的存活率均降低,差异有统计学意义(P<0.05)。与相同浓度亚砷酸钠染毒12 h比较,20.00、30.00μmol/L亚砷酸钠染毒24 h及10.00~30.00μmol/L亚砷酸钠染毒48 h淋巴细胞的存活率均降低,差异有统计学意义(P<0.05)。与对照组相比,10.00~30.00μmol/L亚砷酸钠染毒12 h组和各浓度亚砷酸钠染毒24 h组及20.00、30.00μmol/L亚砷酸钠染毒48 h组淋巴细胞分泌的IL-6水平均较高,差异有统计学意义(P<0.05)。与对照组相比,30.00μmol/L亚砷酸钠染毒12 h组和20.00、30.00μmol/L亚砷酸钠染毒24 h组及10.00~30.00μmol/L亚砷酸钠染毒48 h组淋巴细胞分泌的IL-10水平均较高,差异有统计学意义(P<0.05)。与对照组相比,10.00~30.00μmol/L亚砷酸钠染毒12、24 h组及各浓度亚砷酸钠染毒48 h组淋巴细胞分泌的PFP水平均较高,差异有统计学意义(P<0.05)。与相同浓度亚砷酸钠染毒12 h比较,各浓度亚砷酸钠染毒组淋巴细胞分泌的PFP水平均升高,除10.00μmol/L亚砷酸钠染毒24 h外,差异均有统计学意义(P<0.05)。与对照组相比,30.00μmol/L亚砷酸钠染毒12 h组和20.00、30.00μmol/L亚砷酸钠染毒24、48 h组淋巴细胞分泌的Gr B水平均较高,差异有统计学意义(P<0.05)。不同浓度亚砷酸钠染毒12、24、48 h后淋巴细胞分泌的IL-6、IL-10、Gr B水平间比较,差异均无统计学意义(P>0.05)。结论砷暴露可诱导IL-6、IL-10、穿孔素及颗粒酶B表达水平升高,导致细胞因子分泌平衡紊乱,此可能在砷致免疫系统损伤中发挥重要作用。
Objective To establish human lymphocytes model exposed to sodium arsenite and to understand the effects of sodium arsenite on the secretion of interleukin-6(IL-6), interleukin-10(IL-10), perforin and granzyme B. Methods The venous blood samples were collected from healthy adults and the lymphocytes were isolated by density gradient centrifugation. The lymphocytes were exposed to sodium arsenite at 0.00, 5.00, 10.00, 20.00 and 30.00 μmol/L,respectively. Each group was treated for 12 h, 24 h and 48 h respectively. The arsenic exposed-lymphocytes model was identified by HE staining.Cytotoxicity was detected by MTT assay. ELISA assay was used to detect the levels of IL-6, IL-10, perforin and granzyme B.Results Compared with the control group(0 μmol/L) at different periods, lymphocyte proliferation activity increased in the low dose group(5 μmol/L),while the activities in the groups(10.00-30.00 μmol/L) decreased significantly(P<0.05). Compared with the group exposed to the same concentration of sodium arsenite for 12 h,the activities of lymphocytes in the groups(20.00-30.00 μmol/L) exposed for 24 h and in the group(10.00-30.00 μmol/L) exposed for 48 h were all decreased significantly(P<0.05). Compared with the control group,the expression levels of IL-6 increased significantly in the groups(10.00-30.00 μmol/L)after 12 h-exposure. After 48 h-exposure,IL-6 levels increased significantly in the groups(20.00-30.00 μmol/L)(P<0.05).Compared with the control group, the expression levels of IL-10 increased significantly in the group(30.00 μmol/L) exposed for12 h, the groups(20.00-30.00 μmol/L) exposed for 24 h and the groups(10.00-30.00 μmol/L) exposed for 48 h(P<0.05).Compared with the same dose group exposed for 12 h,the levels of PFP secreted by lymphocytes in each group increased significantly,except for the group of 10.00 μmol/L for 24 h(P<0.05). Compared with the control group,after 12 h-exposure,the levels of Gr B increased significantly in the 30.00 μmol/L group,while after 24 h-and 48 h-exposure,the levels increased significantly in the groups(20.00-30.00 μmol/L)(P <0.05). The levels of IL-6,IL-10 and Gr B secreted by lymphocytes in different dose groups exposed for 12,24,48 h were not statistically different(P <0.05). Conclusion Arsenic exposure can induce the increase of IL-6,IL-10,perforin and granzyme B expression,leading to cytokines secretion disorder subsequently,which may be involved in immune system injury caused by arsenic.
Objective To establish human lymphocytes model exposed to sodium arsenite and to understand the effects of sodium arsenite on the secretion of interleukin-6(IL-6), interleukin-10(IL-10), perforin and granzyme B. Methods The venous blood samples were collected from healthy adults and the lymphocytes were isolated by density gradient centrifugation. The lymphocytes were exposed to sodium arsenite at 0.00, 5.00, 10.00, 20.00 and 30.00 μmol/L,respectively. Each group was treated for 12 h, 24 h and 48 h respectively. The arsenic exposed-lymphocytes model was identified by HE staining.Cytotoxicity was detected by MTT assay. ELISA assay was used to detect the levels of IL-6, IL-10, perforin and granzyme B.Results Compared with the control group(0 μmol/L) at different periods, lymphocyte proliferation activity increased in the low dose group(5 μmol/L),while the activities in the groups(10.00-30.00 μmol/L) decreased significantly(P<0.05). Compared with the group exposed to the same concentration of sodium arsenite for 12 h,the activities of lymphocytes in the groups(20.00-30.00 μmol/L) exposed for 24 h and in the group(10.00-30.00 μmol/L) exposed for 48 h were all decreased significantly(P<0.05). Compared with the control group,the expression levels of IL-6 increased significantly in the groups(10.00-30.00 μmol/L)after 12 h-exposure. After 48 h-exposure,IL-6 levels increased significantly in the groups(20.00-30.00 μmol/L)(P<0.05).Compared with the control group, the expression levels of IL-10 increased significantly in the group(30.00 μmol/L) exposed for12 h, the groups(20.00-30.00 μmol/L) exposed for 24 h and the groups(10.00-30.00 μmol/L) exposed for 48 h(P<0.05).Compared with the same dose group exposed for 12 h,the levels of PFP secreted by lymphocytes in each group increased significantly,except for the group of 10.00 μmol/L for 24 h(P<0.05). Compared with the control group,after 12 h-exposure,the levels of Gr B increased significantly in the 30.00 μmol/L group,while after 24 h-and 48 h-exposure,the levels increased significantly in the groups(20.00-30.00 μmol/L)(P <0.05). The levels of IL-6,IL-10 and Gr B secreted by lymphocytes in different dose groups exposed for 12,24,48 h were not statistically different(P <0.05). Conclusion Arsenic exposure can induce the increase of IL-6,IL-10,perforin and granzyme B expression,leading to cytokines secretion disorder subsequently,which may be involved in immune system injury caused by arsenic.
引文
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[12]刘永莲,张爱华,王大朋,等.调节性T细胞、辅助性T细胞-17相关免疫因子在砷暴露大鼠外周血中的表达差异及意义[J].中华地方病学杂志,2017,36(1):11-15.
[2]于云江,王菲菲,房吉敦,等.环境砷污染对人体健康影响的研究进展[J].环境与健康杂志,2007,24(3):181-183.
[3]Hurst SM,Wilkinson TS,Mcloughlin RM,et al.IL-6 and its soluble receptor orchestrate a temporal switch in the pattern of leukocyte recruitment seen during acute inflammation[J].Immunity,2001,14:705-714.
[4]Song J,Su W,Chen X,et al.Micro RNA-98 suppresses interleukin-10 in peripheral B cells in patient post-cardio transplantation[J].Oncotarget,2017,8:28237-28246.
[5]Sanchez-Muoz F,Dominguez-Lopez A,Yamamoto-Furusho JK.Role of cytokines in inflammatory bowel disease[J].World J Gastroenterol,2008,14:4280-4288.
[6]王璇.穿孔素与颗粒酶B对诊断鼻NK/T细胞淋巴瘤的价值[J].中国卫生标准管理,2016,7(4):169-170.
[7]刘婧,古力娜尔·库尔班,热沙来提·艾米多,等.CD8+T淋巴细胞、颗粒酶B在人类病毒性心肌炎细胞凋亡中的作用[J].热带医学杂志,2013,13(10):1199-1201.
[8]唐波,张捷.穿孔素和颗粒酶B在移植排斥中的研究进展[J].医学综述,2009,15(8):1133-1135.
[9]马艳,孔席丽,张杰,等.亚砷酸钠对体外培养淋巴细胞存活率及金属硫蛋白含量的影响[J].新疆医科大学学报,2008,31(2):148-150.
[10]Khandare AV,Bobade D,Deval M,et al.Expression of negative immune regulatory molecules,pro-inflammatory chemokine and cytokines in immunopathology of ECM developing mice[J].Acta Tropica,2017,172:58-63.
[11]Ma Y,Ren Y,Dai ZJ,et al.IL-6,IL-8 and TNF-αlevels correlate with disease stage in breast cancer patients[J].Adv Clin Exp Med,2017,26:421-426.
[12]刘永莲,张爱华,王大朋,等.调节性T细胞、辅助性T细胞-17相关免疫因子在砷暴露大鼠外周血中的表达差异及意义[J].中华地方病学杂志,2017,36(1):11-15.