Notch信号在大鼠肝纤维化中的作用及常山酮干预机制的研究

发布时间：2018-06-29 06:19

本文选题：Notch + TGF-β　；参考：《青岛大学》2017年硕士论文

【摘要】：目的研究Notch和TGF-β信号通路在肝纤维化大鼠中的相互作用以及对Th17/Treg平衡的影响;探究常山酮干预肝纤维化大鼠,Notch和TGF-β通路的变化及其抗纤维化机制。方法Wistar雄性大鼠随机分为对照组(12只)、模型组(3组,每组12只)。刀豆蛋白A(ConA)建模,模型组大鼠每周尾静脉注射ConA 12.5mg/kg 1次,对照组大鼠每周尾静脉注射磷酸盐缓冲液(PBS)300μl 1次,连续8周;血清生化检测肝功能情况,HE染色观察肝组织病理变化。建模成功后模型组随机分成3组(DAPT组、TGF-β抑制组、DMSO对照组),每组12只,对其体外心尖无菌采血3 ml,分离外周单个核细胞(PBMCs),分别用Notch抑制剂DAPT及TGF-β抑制剂干预培养24 h,RT-PCR测Notch和TGF-β信号mRNA表达,Western Blot测Notch和TGF-β信号相关蛋白表达情况;流式细胞术检测Th17和Treg细胞频率,ELISA检测PBMCs培养上清细胞因子IL-17、IL-23、TGF-β及IL-10的表达水平;Wistar雄性大鼠随机分成模型组、常山酮组和正常组(每组12只),同上ConA建模,常山酮组自造模起灌胃常山酮(10mg/kg),模型组和正常组灌胃等量PBS,连续8周后RT-PCR测Notch和TGF-β信号m RNA表达,Western Blot测Notch和TGF-β信号蛋白表达情况,免疫组化观察肝脏Notch和TGF-β信号表达情况。结果与正常组相比,模型组大鼠的ALT及AST水平显著升高(P0.05),ALB水平显著下降(P0.05),HE染色显示肝细胞坏死伴假小叶形成;模型组Notch和TGF-β信号激活,其中Notch信号的Notch1、Hes1、Hes5和TGF-β信号的TGF-β1和Smad3 mRNA表达显著升高(P0.05),相应的蛋白表达也明显上升(P0.05)。与DMSO组相比,DAPT组Notch和TGF-β信号mRNA表达降低(P0.05),蛋白表达也显著减低(P0.05);TGF-β抑制组Notch和TGF-β信号相关mRNA表达降低(P0.05),蛋白表达亦明显减低(P0.05)。流式及ELISA法检测显示,与DMSO组相比,DAPT组和TGF-β抑制组中Th17细胞频率及其相关细胞因子(IL-17、IL-23)水平降低(P0.05),Treg细胞频率及其相关细胞因子(TGF-β、IL-10)水平亦明显降低(P0.05),Th17/Treg比值均较DMSO组上升。与模型组相比,常山酮组Notch和TGF-β信号Notch1,Hes1,Hes5及TGF-β1,Smad3 mRNA表达显著降低(P0.05);相应蛋白表达也减低(P0.05),与正常组相比无明显差异;免疫组化观察肝组织,常山酮组中Notch1,Hes1,Hes5及TGF-β,Smad3表达较模型组下降(P0.05),与正常组无明显差异。结论Notch和TGF-β信号参与肝纤维化的发生发展,阻断Notch信号能抑制TGF-β信号,抑制TGF-β信号也可阻断Notch信号;Notch和TGF-β信号阻断后可抑制Treg细胞功能,降低TGF-β1水平,改变Th17/Treg细胞平衡;常山酮可抑制Notch和TGF-β信号通路,通过降低Smad3分子表达发挥抗大鼠肝纤维化作用。
[Abstract]:Objective to study the interaction of Notch and TGF- 尾 signaling pathway in rats with hepatic fibrosis and its effect on Th17 / Treg balance, and to explore the changes of Notch and TGF- 尾 pathway and the mechanism of anti-fibrosis in rats with hepatic fibrosis induced by Changshanone. Methods Wistar male rats were randomly divided into control group (n = 12) and model group (n = 12). Concanavalin A (Cona) was established. Cona 12.5mg/kg was injected once a week in the model group and once a week in the control group. After the model was successfully established, the model group was randomly divided into three groups (DAPT group, TGF- 尾 inhibition group, DMSO control group) with 12 rats in each group. Peripheral mononuclear cells (PBMCs) were isolated from the isolated peripheral mononuclear cells (PBMCs) in vitro. Notch inhibitor (DAPT) and TGF- 尾 inhibitor (TGF- 尾 inhibitor) were used to detect Notch and TGF- 尾 signaling mRNA expression by RT-PCR and Western Blot to detect Notch and TGF- 尾 signal-related protein expression. TGF- 尾 and IL-10 expression levels in supernatant of PBMCs cultured by flow cytometry, Th17 and Treg cell frequency were determined by Elisa. Male Wistar rats were randomly divided into three groups: model group, Changshanone group and normal group (12 rats in each group). Cona was used to model the expression of IL-17, IL-23, TGF- 尾 and IL-10 in the supernatant of PBMCs. After 8 weeks, the expression of Notch and TGF- 尾 signal mRNA was measured by RT-PCR. The expression of Notch and TGF- 尾 signal protein was detected by Western blot, and the expression of Notch and TGF- 尾 signal in liver was observed by immunohistochemistry. Results compared with the normal group, the alt and AST levels in the model group were significantly increased (P0.05) and the ALB levels were significantly decreased (P0.05). The expression of TGF- 尾 1 and Smad3 mRNA in Notch Hes1Hes1Hes5 and TGF- 尾 signal was significantly increased (P0.05), and the corresponding protein expression was also significantly increased (P0.05). Compared with DMSO group, Notch and TGF- 尾 signal mRNA expression in DAPT group was decreased (P0.05), protein expression was also significantly decreased (P0.05), Notch and TGF- 尾 signal-related mRNA expression was decreased in TGF- 尾 inhibition group (P0.05), protein expression was also significantly decreased (P0.05). Compared with DMSO group and TGF- 尾 group, Th17 cell frequency and IL-17 IL-23 level in DAPT group and TGF- 尾 inhibitory group were significantly lower than those in DMSO group (P0.05), and the level of TGF- 尾 -IL-10 was significantly lower than that in DMSO group (P0.05) and the ratio of Th17 / Treg was significantly higher than that in DMSO group. Compared with the model group, Notch and TGF- 尾 signal Notch and TGF- 尾 signal Notch1Hes5 and TGF- 尾 1TGF- 尾 1 Smad3 mRNA expression in Changshanone group were significantly decreased (P0.05), the corresponding protein expression was also decreased (P0.05), and there was no significant difference compared with the normal group. The expression of Notch1Hes1Hes5 and TGF- 尾 Smad3 in Changshan ketone group was lower than that in the model group (P0.05), but there was no significant difference between the two groups. Conclusion Notch and TGF- 尾 signal play an important role in the development of hepatic fibrosis. Blocking Notch signal can inhibit TGF- 尾 signal, inhibit TGF- 尾 signal and inhibit TGF- 尾 signal. After blocking Notch signal, Notch and TGF- 尾 signal can inhibit the function of Treg cells, decrease the level of TGF- 尾 1 and change the balance of Th17% Treg cells. Changshanone can inhibit Notch and TGF- 尾 signaling pathway and inhibit the expression of Smad3 molecule.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575.2

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