摘要
目的探讨功能未知基因C6orf120缺失对大鼠自身免疫性肝炎的影响及机制。方法将16只野生型(wild-type,WT)大鼠和16只C6orf120基因敲除型(C6orf120-/-)大鼠分别编号后,采用单纯随机抽样法分别分为2组,每组WT大鼠和C6orf120-/-大鼠各8只。其中一组将刀豆蛋白A(concanavalin A,Con A)以35 mg/kg剂量静脉注射,建立Con A诱导的自身免疫性肝炎大鼠模型,24 h后与另一组未造模的大鼠同时处死,检测肝脏血清学生物化学指标[丙氨酸氨基转移酶(alanine aminotransferase,ALT)和天门冬氨酸氨基转移酶(aspartate aminotransferase,AST)],采用流式细胞术分析WT和C6orf120-/-大鼠的外周血、肝、脾和肠系膜淋巴结内淋巴细胞中CD4+CD25+Foxp3+Treg和CD3+CD8-IL-17A+Th17细胞表达频率的差异。结果 Con A诱导24 h后,C6orf120-/-大鼠血清ALT、AST水平显著低于WT大鼠[ALT:(184.68±48.39)U/L vs (374.84±45.38)U/L,t=8.11,P <0.001;AST:(513.00±80.40)U/L vs(1256.58±27.77)U/L,t=24.72,P <0.001]。Con A诱导24 h后,C6orf120-/-大鼠外周血、肝脏、脾脏、淋巴结内淋巴细胞中Treg的频率分别为(11.70±1.12)%、(12.27±1.25)%、(11.06±1.02)%、(13.96±0.83)%,WT大鼠分别为(9.75±1.01)%、(8.97±0.19)%、(11.80±1.72)%、(14.08±3.15)%,C6orf120-/-大鼠外周血和肝脏中Treg频率显著高于WT大鼠(t=-3.65,P=0.003;t=-6.83,P <0.001),脾脏和淋巴结中Treg频率差异无统计学意义(t值分别为1.05、0.10,P值分别为0.313、0.921)。Con A诱导24 h后,C6orf120-/-大鼠外周血、肝脏、脾脏、淋巴结内淋巴细胞中Th17细胞的频率分别为(0.52±0.08)%、(4.12±0.77)%、(0.78±0.06)%、(0.84±0.04)%,WT大鼠分别为(0.49±0.05)%、(7.74±0.49)%、(1.18±0.06)%、(1.35±0.22)%,C6orf120-/-大鼠和WT大鼠外周血中Th17细胞频率差异无统计学意义(t=-1.06,P=0.309),C6orf120-/-大鼠肝脏、脾脏、淋巴结内淋巴细胞中Th17细胞频率显著低于WT大鼠(t值分别为11.22、12.75、6.52,P均<0.001)。结论 Con A诱导后,C6orf120-/-大鼠外周血和肝脏内淋巴细胞中Treg频率显著上调,肝脏、脾脏和淋巴结内淋巴细胞中Th17频率显著下调,提示C6orf120基因缺失对Con A诱导的大鼠免疫性肝损伤有保护作用,可能与C6orf120基因对Treg和Th17细胞间平衡稳态的免疫调节作用有关。
Objective To investigate the effects of deletion of unknown function gene C6 orf120 on the autoimmune hepatitis of rats and the underlying mechanism. Methods Wild type(WT) rats and C6 orf120 knocked-out(C6 orf120-/-) rats were numbered and divided into 2 groups by simple random sampling method, respectively, 8 WT rats and 8 C6 orf120-/-rats were allocated into each group. Autoimmune hepatitis model was established with Con A 35 mg/kg intravenous injection in rats of one group, which were sacrificed at 24 h after Con A challenging. And the other group were sacrificed at the same time. The severity of liver injury in rats was evaluated by serological examination including serum levels of alanine aminotransferase(ALT) and aspartate aminotransferase(AST). The frequency changes of CD4+CD25+Foxp3+ Treg cells and CD3+ CD8-IL-17 A+ Th17 cells in peripheral blood mononuclear cells, intrahepatic lymphocytes, splenocytes and lymph nodes of WT rats and C6 orf120-/-rats during the course of liver injury were analyzed by flow cytometry, respectively. Results After Con A challenged for 24 h, the serum levels of ALT and AST of C6 orf120-/-rats were significantly lower than those of WT rats [ALT:(184.68 ± 48.39) U/L vs(374.84 ± 45.38) U/L, t = 8.11, P < 0.001;AST:(513.00 ± 80.40) U/L vs(1256.58 ± 27.77) U/L, t = 24.72, P < 0.001]. After Con A challenged for 24 h, the frequencies of Treg cells in peripheral blood mononuclear cells, intrahepatic lymphocytes, splenocytes and lymph nodes in C6 orf120-/-rats were(11.70 ± 1.12)%,(12.27 ± 1.25)%,(11.06 ± 1.02)% and(13.96 ± 0.83)%, respectively, which were(9.75 ± 1.01)%,(8.97 ± 0.19)%,(11.80 ± 1.72)% and(14.08 ± 3.15)% of WT rats, respectively. The frequencies of Treg cells in peripheral blood and intrahepatic lymphocytes of C6 orf120-/-rats were significantly higher than those of WT rats(t =-3.65, P = 0.003;t =-6.83, P < 0.001), there were no significant difference in the frequencies of Treg cells in the splenocytes and lymph nodes(t = 1.05, 0.10;P = 0.313, 0.921). After Con A challenged for 24 h, the frequencies of Th17 cells in peripheral blood mononuclear cells, intrahepatic lymphocytes, splenocytes and lymph nodes in C6 orf120-/-rats were(0.52 ± 0.08)%,(4.12 ± 0.77)%,(0.78 ± 0.06)% and(0.84 ± 0.04)%, respectively, which were(0.49 ± 0.05)%,(7.74 ± 0.49)%,(1.18 ± 0.06)% and(1.35 ± 0.22)% of WT rats, respectively. The frequencies of Th17 cells in peripheral blood between C6 orf120-/-and WT rats(t =-1.06, P = 0.309), the frequencies of Th17 cells in the intrahepatic lymphocytes, splenocytes and lymph nodes of C6 orf120-/-rats were significantly lower than those of WT rats were not significantly different(t = 11.22, 12.75, 6.52;all P < 0.001). Conclusions After Con A induction, and the frequencies of Treg cells in peripheral blood and lymph nodes of C6 orf120-/-rats were significantly up-regulated, and the frequencies of Th17 cells in intrahepatic lymphocytes, splenocytes and lymph nodes were significantly down-regulated. The results indicated that C6 orf120 gene deletion may protect against experimental autoimmune hepatitis induced by Con A, which may be related to the immunoregulatory effect of gene C6 orf120 deletion on the balance of Treg and Th17 cells.
作者
宋心成
张健
吴苑妮
张瑞
张曼卡
马慧敏
李鑫
SONG Xin-cheng;ZHANG Jian;WU Yuan-ni;ZHANG Rui;ZHANG Man-ka;MA Hui-min;LI Xin(Peking University Ditan Teaching Hospital,Beijing 100015,China;Department of Center of Integrated,Traditional Chinese and Western Medicine,Beijing Ditan Hospital,Capital Medical University,Beijing 100015,China;Intensive Care Unit,Aerospace Central Hospital,Beijing 100049,China;Chinese Academy of Medical Sciences&Peking Union Medical College Hospital,Beijing 100730,China)
出处
《中国肝脏病杂志（电子版）》
CAS
2019年第4期42-49,共8页
Chinese Journal of Liver Diseases：Electronic Version
基金
“十三五”国家科技重大专项（2017ZX10205501-001-002）
北京市中医药科技发展资金项目（JJ2015-72）
北京市医院管理局临床医学发展专项经费资助（ZYLX201707）
