脾切除通过促进MDSCs极化对ConA诱导的小鼠肝纤维化的治疗作用及机制
发布时间：2018-09-12 20:33
【摘要】：目的:通过尾静脉注射ConA建立小鼠免疫性肝纤维化模型,造模成功后行脾切除术,研究脾切除对ConA诱导的免疫性肝纤维化小鼠的治疗作用;进一步探讨脾切除能否增强免疫抑制剂对ConA诱导的免疫性肝纤维化小鼠的治疗作用。研究脾切除对髓系抑制细胞(Myeloid-derived suppressor cells,MDSCs)极化、NF-κB信号通路活化及巨噬细胞分型的相关细胞因子表达的影响,初步探讨脾切除对ConA诱导的免疫性肝纤维化小鼠的治疗机制。方法:1、将Balb/c小鼠分为空白组和实验组,实验组通过尾静脉注射刀豆蛋白建立ConA诱导的免疫性肝纤维化模型,剂量为12.5mg/kg,每周一次,连续5周;造模成功后,将实验组小鼠分为ConA造模组、脾切除干预组、假手术组、激素治疗组及脾切除联合激素组,经尾静脉继续注射ConA至第7周。2、分别于造模成功后、ConA注射第7周后,取各组小鼠外周血检测血清丙氨酸转氨酶(Alanine transaminase,ALT)和天门冬氨酸氨基转移酶(Aspartate aminotransferase,AST);通过HE染色评价小鼠肝脏组织炎症程度,Masson染色评估肝脏纤维化程度,免疫组化检测小鼠肝脏组织中平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)的表达水平。3、ConA注射第7周后,取外周血进行流式分析,检测MDSCs极化分型及单核细胞的比例;应用Western blot(WB)方法检测小鼠肝脏NF-κB p50和NF-κB p65转录因子的表达水平;通过免疫荧光方法检测各组小鼠肝脏组织中巨噬细胞的极化分型,应用Real-time PCR方法检测小鼠肝脏组织中M2型巨噬细胞细胞因子及相关炎症因子Arg-1、IL-4及IL-10的表达水平。结果:1、与空白对照组小鼠相比,造模组小鼠血清ALT、AST水平显著升高(p0.05),小鼠肝脏汇管区有较多的炎症细胞浸润,肝脏组织中α-SMA表达水平显著升高(p0.05),伴有轻度肝纤维化。2、ConA连续注射7周后,与ConA造模组相比,激素治疗组、脾切除干预组及脾切除联合激素组小鼠血清ALT、AST水平显著降低(p0.05),肝脏汇管区炎症程度减轻,肝脏组织中α-SMA表达水平显著降低(p0.05),肝脏纤维化程度减轻。与激素治疗组小鼠相比,脾切除联合激素组小鼠肝脏纤维化程度改善更显著(p0.05)。3、与ConA造模组相比,脾切除干预组、激素治疗组及脾切除联合激素组小鼠外周血中CD11b+Ly6ChighMDSCs比例显著增加(P0.05);而脾切除干预组、激素治疗组及脾切除联合激素组小鼠外周血中单核细胞比例较ConA造模组显著减少(P0.05),F4/80+CD206+单核细胞数量有增加趋势。4、脾切除干预组、激素治疗组及脾切除联合激素组小鼠肝脏组织中NF-κB p65转录因子表达量较ConA造模组显著减少(P0.05),而NF-κB p50转录因子表达量较ConA造模组显著增加(P0.05)。5、脾切除干预组、激素治疗组及脾切除联合激素组小鼠肝脏组织中的F4/80+巨噬细胞和F4/80~+iNOS~+M1型巨噬细胞比例较ConA造模组显著减少(P0.05)。而脾切除干预组、激素治疗组及脾切除联合激素组小鼠肝脏组织中ARG-1、IL-4及IL-10的表达量较ConA造模组小鼠显著增加(P0.05)。结论:1、经尾静脉注射ConA 5周可建立Balb/c小鼠免疫性肝纤维化模型。2、脾切除能够改善ConA诱导的免疫性肝损伤小鼠肝脏组织炎症程度,抑制肝星状细胞的活化,延缓肝脏纤维化的进展;脾切除联合激素治疗小鼠免疫性肝纤维化时,两者可能存在协同作用。3、脾切除能够促进外周血中CD11b+Ly6ChighMDSCs的分化,抑制外周血中单核细胞的增殖,并促进F4/80+CD206+单核细胞分化,从而延缓ConA诱导的免疫性肝损伤小鼠肝纤维化的进展。4、脾切除可能通过下调NF-κB p65转录因子表达,同时上调NF-κB p50转录因子表达,从而减轻ConA诱导的小鼠免疫性肝纤维化。5、脾切除能够明显降低肝脏组织中F4/80+巨噬细胞和F4/80~+iNOS~+M1型巨噬细胞的比例,并诱导M2型巨噬细胞相关细胞因子及炎症因子的表达,从而减轻ConA诱导的免疫性肝损伤小鼠的肝脏纤维化。6、脾切除可能通过抑制NF-κB信号通路活化,促进MDSCs转化为M2型巨噬细胞,从而减轻ConA诱导的肝损伤,延缓肝纤维化的进展。
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575.2
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575.2
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