【摘要】：目的:通過尾靜脈注射ConA建立小鼠免疫性肝纖維化模型,造模成功后行脾切除術(shù),研究脾切除對ConA誘導(dǎo)的免疫性肝纖維化小鼠的治療作用;進(jìn)一步探討脾切除能否增強(qiáng)免疫抑制劑對ConA誘導(dǎo)的免疫性肝纖維化小鼠的治療作用。研究脾切除對髓系抑制細(xì)胞(Myeloid-derived suppressor cells,MDSCs)極化、NF-κB信號通路活化及巨噬細(xì)胞分型的相關(guān)細(xì)胞因子表達(dá)的影響,初步探討脾切除對ConA誘導(dǎo)的免疫性肝纖維化小鼠的治療機(jī)制。方法:1、將Balb/c小鼠分為空白組和實(shí)驗(yàn)組,實(shí)驗(yàn)組通過尾靜脈注射刀豆蛋白建立ConA誘導(dǎo)的免疫性肝纖維化模型,劑量為12.5mg/kg,每周一次,連續(xù)5周;造模成功后,將實(shí)驗(yàn)組小鼠分為ConA造模組、脾切除干預(yù)組、假手術(shù)組、激素治療組及脾切除聯(lián)合激素組,經(jīng)尾靜脈繼續(xù)注射ConA至第7周。2、分別于造模成功后、ConA注射第7周后,取各組小鼠外周血檢測血清丙氨酸轉(zhuǎn)氨酶(Alanine transaminase,ALT)和天門冬氨酸氨基轉(zhuǎn)移酶(Aspartate aminotransferase,AST);通過HE染色評價(jià)小鼠肝臟組織炎癥程度,Masson染色評估肝臟纖維化程度,免疫組化檢測小鼠肝臟組織中平滑肌肌動(dòng)蛋白(α-smooth muscle actin,α-SMA)的表達(dá)水平。3、ConA注射第7周后,取外周血進(jìn)行流式分析,檢測MDSCs極化分型及單核細(xì)胞的比例;應(yīng)用Western blot(WB)方法檢測小鼠肝臟NF-κB p50和NF-κB p65轉(zhuǎn)錄因子的表達(dá)水平;通過免疫熒光方法檢測各組小鼠肝臟組織中巨噬細(xì)胞的極化分型,應(yīng)用Real-time PCR方法檢測小鼠肝臟組織中M2型巨噬細(xì)胞細(xì)胞因子及相關(guān)炎癥因子Arg-1、IL-4及IL-10的表達(dá)水平。結(jié)果:1、與空白對照組小鼠相比,造模組小鼠血清ALT、AST水平顯著升高(p0.05),小鼠肝臟匯管區(qū)有較多的炎癥細(xì)胞浸潤,肝臟組織中α-SMA表達(dá)水平顯著升高(p0.05),伴有輕度肝纖維化。2、ConA連續(xù)注射7周后,與ConA造模組相比,激素治療組、脾切除干預(yù)組及脾切除聯(lián)合激素組小鼠血清ALT、AST水平顯著降低(p0.05),肝臟匯管區(qū)炎癥程度減輕,肝臟組織中α-SMA表達(dá)水平顯著降低(p0.05),肝臟纖維化程度減輕。與激素治療組小鼠相比,脾切除聯(lián)合激素組小鼠肝臟纖維化程度改善更顯著(p0.05)。3、與ConA造模組相比,脾切除干預(yù)組、激素治療組及脾切除聯(lián)合激素組小鼠外周血中CD11b+Ly6ChighMDSCs比例顯著增加(P0.05);而脾切除干預(yù)組、激素治療組及脾切除聯(lián)合激素組小鼠外周血中單核細(xì)胞比例較ConA造模組顯著減少(P0.05),F4/80+CD206+單核細(xì)胞數(shù)量有增加趨勢。4、脾切除干預(yù)組、激素治療組及脾切除聯(lián)合激素組小鼠肝臟組織中NF-κB p65轉(zhuǎn)錄因子表達(dá)量較ConA造模組顯著減少(P0.05),而NF-κB p50轉(zhuǎn)錄因子表達(dá)量較ConA造模組顯著增加(P0.05)。5、脾切除干預(yù)組、激素治療組及脾切除聯(lián)合激素組小鼠肝臟組織中的F4/80+巨噬細(xì)胞和F4/80~+iNOS~+M1型巨噬細(xì)胞比例較ConA造模組顯著減少(P0.05)。而脾切除干預(yù)組、激素治療組及脾切除聯(lián)合激素組小鼠肝臟組織中ARG-1、IL-4及IL-10的表達(dá)量較ConA造模組小鼠顯著增加(P0.05)。結(jié)論:1、經(jīng)尾靜脈注射ConA 5周可建立Balb/c小鼠免疫性肝纖維化模型。2、脾切除能夠改善ConA誘導(dǎo)的免疫性肝損傷小鼠肝臟組織炎癥程度,抑制肝星狀細(xì)胞的活化,延緩肝臟纖維化的進(jìn)展;脾切除聯(lián)合激素治療小鼠免疫性肝纖維化時(shí),兩者可能存在協(xié)同作用。3、脾切除能夠促進(jìn)外周血中CD11b+Ly6ChighMDSCs的分化,抑制外周血中單核細(xì)胞的增殖,并促進(jìn)F4/80+CD206+單核細(xì)胞分化,從而延緩ConA誘導(dǎo)的免疫性肝損傷小鼠肝纖維化的進(jìn)展。4、脾切除可能通過下調(diào)NF-κB p65轉(zhuǎn)錄因子表達(dá),同時(shí)上調(diào)NF-κB p50轉(zhuǎn)錄因子表達(dá),從而減輕ConA誘導(dǎo)的小鼠免疫性肝纖維化。5、脾切除能夠明顯降低肝臟組織中F4/80+巨噬細(xì)胞和F4/80~+iNOS~+M1型巨噬細(xì)胞的比例,并誘導(dǎo)M2型巨噬細(xì)胞相關(guān)細(xì)胞因子及炎癥因子的表達(dá),從而減輕ConA誘導(dǎo)的免疫性肝損傷小鼠的肝臟纖維化。6、脾切除可能通過抑制NF-κB信號通路活化,促進(jìn)MDSCs轉(zhuǎn)化為M2型巨噬細(xì)胞,從而減輕ConA誘導(dǎo)的肝損傷,延緩肝纖維化的進(jìn)展。
[Abstract]:AIM: To establish a mouse model of immune hepatic fibrosis by injecting ConA into tail vein and to study the therapeutic effect of splenectomy on ConA-induced immune hepatic fibrosis in mice. In addition to the effects of myeloid-derived suppressor cells (MDSCs) polarization, activation of NF-kappa B signaling pathway and expression of cytokines related to macrophage typing, the therapeutic mechanism of splenectomy on ConA-induced immune hepatic fibrosis in mice was preliminarily investigated. Methods: 1. Balb/c mice were divided into blank group and experimental group. ConA-induced immune hepatic fibrosis model was established by intravenous injection of concanavalin at a dose of 12.5mg/kg once a week for 5 weeks. After 7 weeks of ConA injection, the serum levels of Alanine transaminase (ALT) and Aspartate aminotransferase (AST) were detected in peripheral blood of mice in each group; the degree of inflammation in liver tissue was evaluated by HE staining; the degree of liver fibrosis was evaluated by Masson staining; and the degree of liver fibrosis was detected by immunohistochemistry. The expression level of smooth muscle actin (alpha-SMA) in liver tissue was measured by flow cytometry after 7 weeks of ConA injection. The polarization of MDSCs and the ratio of monocytes were detected by flow cytometry. The expression levels of NF-kappa B P50 and NF-kappa B p65 transcription factors in liver of mice were detected by Western blot (WB). The polarization patterns of macrophages in liver tissues of mice were detected by light method, and the expression levels of M2 macrophage cytokines and related inflammatory factors Arg-1, IL-4 and IL-10 in liver tissues of mice were detected by Real-time PCR. There were more inflammatory cells infiltration in portal area of mice liver, and the expression of alpha-SMA in liver tissue was significantly increased (p0.05), accompanied by mild hepatic fibrosis. 2. After 7 weeks of ConA continuous injection, compared with ConA model group, the levels of ALT and AST in serum of mice in hormone treatment group, splenectomy intervention group and splenectomy combined with hormone group were significantly decreased (p0.05). Compared with the hormone treatment group, the degree of liver fibrosis in the splenectomy combined with hormone group was improved more significantly (p0.05). 3. Compared with the ConA model group, the splenectomy intervention group, the hormone treatment group and the splenectomy combined with hormone group were smaller. The proportion of CD11b+Ly6 ChighMDSCs in peripheral blood of mice increased significantly (P 0.05), while the proportion of monocytes in peripheral blood of mice in splenectomy intervention group, hormone treatment group and splenectomy combined hormone group decreased significantly (P 0.05), and the number of F4/80+CD206+ monocytes increased significantly (P 0.05), splenectomy intervention group, hormone treatment group and splenectomy combined stimulation group. The expression of NF-kappa B p65 transcription factor in liver tissue of vegetable group was significantly lower than that of ConA group (P 0.05), while the expression of NF-kappa B P50 transcription factor was significantly higher than that of ConA group (P 0.05). The expression of F4/80+ macrophages and F4/80~+iNOS~+M1 macrophages in liver tissue of splenectomy intervention group, hormone treatment group and splenectomy combined hormone group was significantly higher than that of ConA group (P 0.05). The expression of ARG-1, IL-4 and IL-10 in liver tissue of splenectomy intervention group, hormone treatment group and splenectomy combined hormone group was significantly higher than that of ConA model group (P 0.05). Conclusion: 1. Immune hepatic fibrosis model of Balb/c mice can be established by intravenous injection of ConA for 5 weeks. Splenectomy combined with hormone therapy may have synergistic effect on immunological liver fibrosis in mice. 3. Splenectomy can promote the differentiation of CD11b + Ly6 ChighMDSCs in peripheral blood and inhibit the development of liver fibrosis. Splenectomy may reduce the expression of NF-kappa B p65 transcription factor and up-regulate the expression of NF-kappa B P50 transcription factor, thereby alleviating ConA-induced immune liver fibrosis in mice. Resection can significantly reduce the proportion of F4/80+ macrophages and F4/80~+iNOS~+M1 macrophages in liver tissue, and induce the expression of macrophage-related cytokines and inflammatory factors in M2, thereby reducing the liver fibrosis in ConA-induced immune liver injury mice. 6. Splenectomy may promote MD by inhibiting the activation of NF-kappa B signaling pathway. SCs can be transformed into M2 macrophages, thereby alleviated ConA induced liver injury and delaying the progression of liver fibrosis.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R575.2

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