本文選題：Notch + TGF-β　； 參考：《青島大學(xué)》2017年碩士論文

【摘要】：目的研究Notch和TGF-β信號(hào)通路在肝纖維化大鼠中的相互作用以及對(duì)Th17/Treg平衡的影響;探究常山酮干預(yù)肝纖維化大鼠,Notch和TGF-β通路的變化及其抗纖維化機(jī)制。方法Wistar雄性大鼠隨機(jī)分為對(duì)照組(12只)、模型組(3組,每組12只)。刀豆蛋白A(ConA)建模,模型組大鼠每周尾靜脈注射ConA 12.5mg/kg 1次,對(duì)照組大鼠每周尾靜脈注射磷酸鹽緩沖液(PBS)300μl 1次,連續(xù)8周;血清生化檢測(cè)肝功能情況,HE染色觀察肝組織病理變化。建模成功后模型組隨機(jī)分成3組(DAPT組、TGF-β抑制組、DMSO對(duì)照組),每組12只,對(duì)其體外心尖無(wú)菌采血3 ml,分離外周單個(gè)核細(xì)胞(PBMCs),分別用Notch抑制劑DAPT及TGF-β抑制劑干預(yù)培養(yǎng)24 h,RT-PCR測(cè)Notch和TGF-β信號(hào)mRNA表達(dá),Western Blot測(cè)Notch和TGF-β信號(hào)相關(guān)蛋白表達(dá)情況;流式細(xì)胞術(shù)檢測(cè)Th17和Treg細(xì)胞頻率,ELISA檢測(cè)PBMCs培養(yǎng)上清細(xì)胞因子IL-17、IL-23、TGF-β及IL-10的表達(dá)水平;Wistar雄性大鼠隨機(jī)分成模型組、常山酮組和正常組(每組12只),同上ConA建模,常山酮組自造模起灌胃常山酮(10mg/kg),模型組和正常組灌胃等量PBS,連續(xù)8周后RT-PCR測(cè)Notch和TGF-β信號(hào)m RNA表達(dá),Western Blot測(cè)Notch和TGF-β信號(hào)蛋白表達(dá)情況,免疫組化觀察肝臟Notch和TGF-β信號(hào)表達(dá)情況。結(jié)果與正常組相比,模型組大鼠的ALT及AST水平顯著升高(P0.05),ALB水平顯著下降(P0.05),HE染色顯示肝細(xì)胞壞死伴假小葉形成;模型組Notch和TGF-β信號(hào)激活,其中Notch信號(hào)的Notch1、Hes1、Hes5和TGF-β信號(hào)的TGF-β1和Smad3 mRNA表達(dá)顯著升高(P0.05),相應(yīng)的蛋白表達(dá)也明顯上升(P0.05)。與DMSO組相比,DAPT組Notch和TGF-β信號(hào)mRNA表達(dá)降低(P0.05),蛋白表達(dá)也顯著減低(P0.05);TGF-β抑制組Notch和TGF-β信號(hào)相關(guān)mRNA表達(dá)降低(P0.05),蛋白表達(dá)亦明顯減低(P0.05)。流式及ELISA法檢測(cè)顯示,與DMSO組相比,DAPT組和TGF-β抑制組中Th17細(xì)胞頻率及其相關(guān)細(xì)胞因子(IL-17、IL-23)水平降低(P0.05),Treg細(xì)胞頻率及其相關(guān)細(xì)胞因子(TGF-β、IL-10)水平亦明顯降低(P0.05),Th17/Treg比值均較DMSO組上升。與模型組相比,常山酮組Notch和TGF-β信號(hào)Notch1,Hes1,Hes5及TGF-β1,Smad3 mRNA表達(dá)顯著降低(P0.05);相應(yīng)蛋白表達(dá)也減低(P0.05),與正常組相比無(wú)明顯差異;免疫組化觀察肝組織,常山酮組中Notch1,Hes1,Hes5及TGF-β,Smad3表達(dá)較模型組下降(P0.05),與正常組無(wú)明顯差異。結(jié)論Notch和TGF-β信號(hào)參與肝纖維化的發(fā)生發(fā)展,阻斷Notch信號(hào)能抑制TGF-β信號(hào),抑制TGF-β信號(hào)也可阻斷Notch信號(hào);Notch和TGF-β信號(hào)阻斷后可抑制Treg細(xì)胞功能,降低TGF-β1水平,改變Th17/Treg細(xì)胞平衡;常山酮可抑制Notch和TGF-β信號(hào)通路,通過(guò)降低Smad3分子表達(dá)發(fā)揮抗大鼠肝纖維化作用。
[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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R575.2

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本文編號(hào)：2081191