【作者】 周瑞；

【导师】 喻红；

【作者基本信息】 武汉大学 ， 生物化学与分子生物学， 2018， 硕士

【摘要】 目的:肾脏是人体重要的排泄器官和内分泌器官,在维持机体内环境方面发挥重要作用。脂代谢紊乱是代谢性疾病的主要危险因素,动脉粥样硬化、脂肪肝和糖尿病等的发生发展都与之密切相关。近年来研究表明,脂代谢紊乱使机体处于慢性炎症和氧化应激状态,进而导致肾脏功能受损。高密度脂蛋白(HDL)作为动脉粥样硬化(As)预防因子而备受关注。近年来研究发现HDL失功能可加剧体内炎症、氧化应激状态,而B族Ⅰ型清道夫受体(SR-BI)是HDL功能性受体,参与机体脂代谢的调节,SR-BI基因缺失会导致小鼠体内血脂紊乱、HDL功能异常及肾组织损伤。甲硫亚砜还原酶A(MsrA)是胞内特异性还原酶,其抗氧化功能对维持体内氧化还原状态平衡至关重要。实验室前期已发现慢病毒Lv-MsrA-GFP注射的小鼠,肝脏、心脏、肾脏等组织中MsrA均有明显升高,且在apoE基因敲除小鼠(ApoE-/-)体内能够通过调控肝脏脂质代谢活动,降低机体血脂水平,改善其炎症及氧化状态,延缓ApoE-/-小鼠As病程。本研究以高脂饲养的SR-BI基因敲除小鼠(SR-BI-/-)为模型,拟通过hMsrA高表达干预机体整体氧还状态而改善HDL功能,观察其改变对肾脏纤维化的影响并探究其相关机制。方法:选取雌性、10-11月龄普食喂养的野生型(WT)与SR-BI-/-小鼠进行基础状态的比较,通过采用生化酶法试剂盒检测小鼠血浆总胆固醇(TC)、游离胆固醇(FC)、甘油三酯(TG);连续动态法测定小鼠血浆对氧磷酶1(PON1)活性;肾脏组织石蜡切片行Masson染色观察肾脏形态学变化以分析小鼠肾脏纤维化状况;qPCR检测两种小鼠肾脏中氧化炎症及纤维化相关因子基因表达,即肿瘤坏死因子α(TNF-α)、IV型胶原(COLIV)、基质金属蛋白酶2(MMP2)、基质金属蛋白酶9(MMP 9)等。利用实验室构建的重组质粒pWPI-GFP,pWPI-GFP-MsrA通过慢病毒包装获得慢病毒颗粒Lv-GFP,Lv-GFP-MsrA。将SR-BI-/-小鼠随机分为两组,每组七只,采取球后静脉注射的方式将慢病毒颗粒导入小鼠体内。普食喂养两周后,更换高脂饲料加速小鼠体内血脂紊乱状态,高脂八个月后,异氟烷麻醉取血,将小鼠安乐死,解剖后组织取材分析。利用qPCR及Western blotting验证MsrA在肾脏高表达;采用生化酶法试剂盒检测小鼠血浆TC、FC、TG;连续动态法测定小鼠PON1活性;肾脏组织石蜡切片行Masson染色观察肾脏形态学变化,观察小鼠肾脏纤维化状况;运用qPCR和Western Blotting检测纤维化相关分子Fn、COL I、COLⅣV、转化生长因子-β(TGF-β)、MMP2、MMP9及炎症氧化相关分子 PON1和IL-1 β,IL-6等mRNA及蛋白的表达。结果:基础状态下,SR-BI-/-小鼠血浆TC,FC含量明显高于野生型小鼠,TG无明显差异,抗氧化蛋白PON1含量明显低于WT小鼠。肾脏masson染色结果显示,与WT小鼠相比,SR-BI-/-肾纤维化较严重(P<0.05)。与WT小鼠相比,SR-BI-/-小鼠MsrA表达降低,TNF-α升高(P<0.01)。肾脏纤维化指标Collagen Ⅳ在SR-BI7-小鼠肾脏中表达高于WT(P<0.05),而降解细胞外基质的蛋白酶MMP 2及MMP 9的表达量在SR-BI-/-小鼠肾脏中的表达量明显低于WT小鼠(P<0.05)。利用慢病毒颗粒Lv-GFP和Lv-GFP-MsrA感染SR-BI-/-小鼠,高脂八月后安乐死。利用qPCR及Western blotting证实肾脏MsrA,结果显示高表达模型成功建立。与LV-GFP组相比,LV-MsrA组血浆中TC、FC及TG水平显著下降(P<0.01);Lv-MsrA组血浆PON1活性明显高于对照组(P<0.01);肾脏石蜡切片切片行Masson染色,结果显示Lv-MsrA组小鼠纤维化度明显减轻;肾脏qPCR检测结果显示,炎症因子IL-1β、IL-6,TNF-αmRNA显著下降(P<0.05),LV-MsrA 组 PON1 表达明显上升(P<0.05),而 Fn、COL1、COLⅣ、TGF-β 等纤维化相关基因mRNA相对表达量与对照组相比明显下降(P<0.05),MMP2、MMP9表达量显著增加(P<0.05)。结论:我们成功构建了 Lv-GFP-MsrA慢病毒载体,通过感染SR-BI-/-小鼠使MsrA在小鼠体内高表达,通过其抗氧化作用,调节体内血脂紊乱,降低机体炎症水平,改善氧化应激状态,减轻肾脏纤维化病变,延缓肾脏损伤进程。更多还原

【Abstract】 Objective:As an important excretory and endocrine organ of human body,the kidney plays an important role in maintaining the internal environment.Lipid metabolism disorder is a major risk factor for metabolic diseases,which related closely to the occurrence and development of atherosclerosis,fatty liver,and diabetes.Recent studies have shown that disorders of lipid metabolism put the body into a state of chronic inflammation and oxidative stress,leading to impaired kidney function.High-density lipoprotein(HDL)has attracted much attention as a prophylactic factor for atherosclerosis(As).In recent years,it was found that HDL dysfunction can exacerbate inflammation and oxidative stress in vivo.However,group B type I scavenger receptors(SR-BI)are functional receptors of HDL and participate in the regulation of lipid metabolism,the deletion of which could cause dyslipidemia,HDL dysfunction and renal tissue damage in mice.Methionyl sulfoxide reductase A(MsrA)is an intracellular-specific reductase,and its antioxidative function is essential to maintain the homeostasis of the redox state in the body.Upon the previous experiments of the laboratory,the level of MsrA was found to have significantly increased in liver,heart,kidney and other tissues of the mice injected with Lv-MsrA-GFP,and can reduce the blood lipid levels,improve the inflammation and oxidation status and delay the progression of AS in ApoE-/-mice.In this study,we used SR-BI-/-mice fed with a high-fat diet as the HDL dysfunctional animal model.Regarding HDL as a regulatory target,we observed its effects in kidney fibrosis and summarized the related mechanisms by over expression of hMsrA interfered with the overall oxygen status of the body to improve the function of HDL.METHODS:Chosen from WT and SR-BI-/-female mice,which were fed to 10-11 months of age,the basal states were compared and the total cholesterol(TC),free cholesterol(FC)and triglyceride(TG)in the plasma were measured by the biochemical enzyme kit;mouse plasma paraoxonase 1(PON1)activity was measured by continuous dynamic assay;kidney morphology changes were observed to analyze renal fibrosis by Masson staining;the expression of oxidative inflammation and fibrosis-related genes in two mouse kidneys,such as tumor necrosis factor alpha(TNF-alpha),type Ⅳcollagen(COL IV),matrix metalloproteinase 2(MMP 2),matrix metalloproteinase 9(MMP 9)and so on,were detected by qPCR.Lv-GFP and Lv-GFP-MsrA,the lentiviral particles,were obtained by lentivirus packaging using the recombinant plasmid pWPI-GFP and pWPI-GFP-MsrA constructed by the laboratory.The SR-BI-/-mice were randomly divided into two groups with each group of seven,and the lentivirus particles were injected to the mice by means of retrobulbar injection.After two weeks of general diet,replacement of a high-fat diet could accelerate the dyslipidemia in the mice.After eight months of high-fat diet,the mice were anesthetized by isoflurane for blood collection and then were dissected to obtain the tissues.The high expression of MsrA in the kidney was verified by qPCR and western blotting;plasma total cholesterol(TC),free cholesterol(FC)and triglyceride(TG)were detected by biochemical enzymatic kit;the continuous dynamic method was used to determine paraoxonase 1(PON1)activity in the plasma;kidney tissue paraffin sections were visualized by Masson staining to observe renal morphological changes and to observe the renal fibrosis in·mice;qPCR and western blotting were used to detect fibrosis-related molecules,such as fibronectin(Fn),type I collagen(COL I),type IV collagen(COL IV),transforming growth factor-(β(TGF-β),matrix metalloproteinase 2(MMP2),matrix metalloproteinase 9(MMP9),and the mRNA and protein expression of inflammatory oxidative-related molecules,such as PON1,IL-1β,IL-6 and so on.Results:Under basal conditions,the content of TC and FC in the plasma of SR-BI-/-mice was significantly higher than that in wild-type mice,while TG had no significant difference,and antioxidant protein PON1 content was significantly lower than that in WT mice.Masson staining showed that the renal fibrosis in SR-BI-/-mice was more severe than that in WT mice(P<0.05).Compared with WT mice,the expression of MsrA was decreased and TNF-a was increased(P<0.01)in SR-BI-/-mice.The expression of Collagen IV(a target of renal fibrosis)was higher in kidneys of SR-BI-/-mice than in WT mice(P<0.05),while the expression levels of proteasome MMP 2 and MMP 9 in extracellular matrix were significantly lower in SR-BI-/-mice than those in WT mice(P<0.05).The SR-BI-/-mice were infected with lentivirus particles Lv-GFP and Lv-GFP-MsrA and euthanized after eight-month high-fat diet.It was confirmed by qPCR and western blotting that renal MsrA was highly expressed and a high expression model was successfully established.Compared with LV-GFP group,TC,FC and TG levels were significantly decreased(P<0.01)and PON]activity was significantly higher(P<0.01)in the plasma of LV-MsrA group.Masson staining showed that the degree of fibrosis was significantly reduced in the kidney of Lv-MsrA group.The results of qPCR showed that the inflammatory cytokines IL-1β,IL-6,and TNF-a mRNA were significantly decreased(P<0.05)in the kidney.The expression of PON1,MMP2 and MMP9 was significantly increased(P<0.05)in Lv-MsrA group,while the relative mRNA expression of Fn,COL 1,COL IV,TGF-β and other fibrosis-related genes were significantly decreased(P<0.05).Conclusion:We have successfully constructed the Lv-GFP-MsrA lentiviral vector and infected SR-BI-/-mice to highly express MsrA in mice.Due to its anti-oxidation,it can regulate the dyslipidemia,reduce the body’s inflammation level,improve the oxidative stress and HDL’s function,extenuate renal fibrotic lesions,and delay the progression of kidney damage.更多还原