重组腺病毒ALR的构建及过表达ALR在软脂酸诱导HepG2细胞脂肪变性中的作用

发布时间：2018-04-08 22:35

本文选题：肝再生增强因子　切入点：腺病毒　出处：《重庆医科大学》2016年硕士论文

【摘要】：背景:近年来非酒精性脂肪肝病(Non-alcoholic fatty liver disease,NAFLD)发病率明显上升,西方发达国家的年发病率高达20%-40%,在中国发病仅次于病毒性肝病,且有极大可能发展至肝硬化、肝癌以及肝功能衰竭,危及生命。报道显示,与普通人群相比,脂肪肝患者的心血管疾病死亡率较高。因此,NAFLD是人类健康的一大严重威胁。且NAFLD的临床表现轻微,无特异性表现,诊断多依赖实验室检查,也无批准的单药治疗方案,因此对NAFLD的发病机制进一步研究以寻找有效的分子标志物用于NAFLD的早期筛查,并发现新的治疗靶点予以针对性治疗,这对提高早期诊断率及改善预后意义重大。肝再生增强因子(augmenter of liver regeneration,ALR),首次发现是定位于肝细胞浆,具有促进细胞增殖、抗细胞凋亡,调节免疫,逆转肝脏纤维化等作用的一种非特异性细胞因子。ALR在体内发现有三种分子片段:15KD、21KD和23KD。15KD是23KD的C′末端片断,即15KD ALR无信号肽序列,目前研究23KD ALR主要是在线粒体功能活动发挥作用。已有研究表明特异性敲除小鼠23KD的ALR基因后,肝脏的脂肪性肝炎及肝癌的发病率明显增加,而且在NAFLD的病人肝组织标本检测中发现23KD ALR的表达较正常人肝是下调的,说明23 KD ALR在NAFLD的发生发展中起重要作用,而15KD ALR作为23KDALR的剪切片段,目前国内外研究中暂无报道其在NAFLD中的生物学作用。因此,本实验主要构建重组15KD ALR腺病毒及探讨过表达ALR对软脂酸诱导Hep G2细胞脂肪变性的作用,为NAFLD的诊治提供新的思路。本研究包括两个部分:第一部分:重组Ad-m ALR和Ad-h ALR的构建和鉴定目的:重组腺病毒含肝再生增强因子(ALR)基因的构建。方法:基因重组法构建重组穿梭载体(p Ad Track-TO4-m ALR和p Ad Track-TO4-h ALR),重组腺病毒(Ad-GFP-m ALR和Ad-GFP-h ALR)采用同源重组法构建,4轮扩增后获得高低度重组腺病毒。感染L02细胞,通过荧光蛋白的表达了解其感染率,ALR蛋白表达情况和其增殖活性则通过Western Blotting法和CCK-8法检测。结果:1.重组腺病毒(Ad-GFP-m ALR和Ad-GFP-h ALR)被构建成功。2.重组腺病毒(Ad-GFP-m ALR和Ad-GFP-h ALR)能高效感染并稳定表达于L02细胞。3.与非感染组和空载病毒组相比,过表达ALR均能促进L02细胞的增殖。结论:成功构建重组腺病毒Ad-GFP-m ALR和Ad-GFP-h ALR,细胞实验证实15KD ALR对L02细胞具有促增殖作用。第二部分:过表达ALR在软脂酸诱导Hep G2细胞脂肪变性中的作用目的:观察过表达15KD ALR在软脂酸(palmitic acid,PA)诱导Hep G2细胞脂肪变性的作用,并简要探讨其机制。方法:本实验分为5组:未感染腺病毒且无PA处理的Hep G2细胞组(G2-NC-No infection),感染空载病毒无PA的Hep G2细胞组(G2-Ad-GFP-NC),感染空载病毒有PA的Hep G2细胞组(G2-Ad-GFP-PA),,感染Ad-GFP-h ALR无PA的Hep G2细胞组(G2-Ad-GFP-h ALR-NC),感染Ad-GFP-h ALR有PA的Hep G2细胞组(G2-Ad-GFP-h ALR-PA)。甘油三酯(Triglyceride,TG)酶法和尼罗红染色法定量和定性检测胞内脂滴储积情况,Real Time-PCR和Western blotting法被应用于细胞内固醇类调节原件结合蛋白-1(SREBP-1),脂肪分化相关蛋白(ADRP)和过氧化物酶体增殖物激活受体-α(PPAR-α)的检测,流式细胞仪检测凋亡率,Western Blotting法检测MAPK通路相关蛋白。结果:1.与对照组G2-NC-No infection、G2-Ad-GFP-NC、G2-Ad-GFP-h ALR-NC相比,实验组G2-Ad-GFP-PA的TG的测定是明显增加的,脂滴的红色荧光强度是显著增强的。而与G2-Ad-GFP-PA组相比,过表达ALR(G2-Ad-GFP-h ALR-PA组)后,细胞内的TG及脂滴的红色荧光强度明显下调。2.与对照组G2-NC-No infection、G2-Ad-GFP-NC、G2-Ad-GFP-h ALR-NC相比,实验组G2-Ad-GFP-PA中SREBP-1、PPAR-α,ADRP的m RNA及蛋白的表达均显著增加。与G2-Ad-GFP-PA组相比,过表达ALR(G2-Ad-GFP-h ALR-PA组)后,胞内SREBP-1、PPAR-α,ADRP的m RNA及蛋白表达较实验组G2-Ad-GFP-PA下调。3.与对照组G2-NC-No infection、G2-Ad-GFP-NC、G2-Ad-GFP-h ALR-NC相比,实验组G2-Ad-GFP-PA中的凋亡率是明显增加的。但与G2-Ad-GFP-PA组比较,过表达ALR(G2-Ad-GFP-h ALR-PA组)后Hep G2细胞的凋亡率明显降低。4.与对照组G2-NC-No infection、G2-Ad-GFP-NC、G2-Ad-GFP-h ALR-NC相比,实验组G2-Ad-GFP-PA中的磷酸化蛋白p-jnk、p-p42/44、p-p38相对表达明显增加。但与G2-Ad-GFP-PA组比较,过表达ALR(G2-Ad-GFP-PA)组中磷酸化蛋白p-jnk、p-p42/44、p-p38相对表达明显下调。结论:1.软脂酸能诱导Hep G2细胞的脂肪变性。2.过表达ALR可减轻PA诱导Hep G2细胞的脂滴储积。3.过表达ALR可降低脂肪合成代谢中关键酶(SREBP-1、PPAR-α、ADRP)的表达.4.过表达ALR可减少PA诱导Hep G2细胞的凋亡的发生。5.过表达ALR可抑制MAPK通路中磷酸化蛋白(p-JNK,p-p42/p44,p-p38)的产生。
[Abstract]:Background: in recent years, nonalcoholic fatty liver disease (Non-alcoholic fatty liver disease, NAFLD) incidence increased significantly, western developed countries, the annual incidence rate of up to 20%-40%, in China disease after viral hepatitis, which may progress to cirrhosis and liver cancer, and liver failure, life-threatening. Reports show that compared with the ordinary the crowd, fatty liver in patients with cardiovascular disease and high mortality rate. Therefore, NAFLD is a serious threat to human health. The clinical manifestations and NAFLD of mild, nonspecific manifestation, diagnosis depends on the laboratory examination, treatment without approval of the single drug, so the pathogenesis of NAFLD for further research to find effective molecular marker for early screening of NAFLD, and find new therapeutic targets to targeted therapy, to improve the early diagnosis rate and improve the prognosis of great significance. Augmenter of liver regeneration (augm Enter of liver regeneration, ALR), first discovered is located in the cytoplasm of hepatocytes, can promote cell proliferation, anti apoptosis, immune regulation, a nonspecific cytokine.ALR reversal of hepatic fibrosis in vivo shows that there are three kinds of molecular fragments: 15KD, 21KD and 23KD.15KD is C 'terminal fragment of 23KD that is, 15KD ALR without signal peptide sequence, the present study 23KD ALR plays a major role in mitochondrial function activities. Studies have shown that specific ALR gene knock in mice after 23KD, liver steatohepatitis and hepatocellular carcinoma was significantly increased, and found that the expression of 23KD ALR was down regulated in normal human liver the detection of NAFLD in liver tissue specimens, 23 KD ALR plays an important role in the development of NAFLD, 15KD and ALR as the shearing segment of 23KDALR, currently there are no research reported in NAFLD biology Use. Therefore, this experiment is to construct recombinant adenovirus 15KD ALR and explore the expression of ALR on palmitic acid induced Hep G2 cell steatosis, to provide new ideas for the diagnosis and treatment of NAFLD. This study includes two parts: the first part: Construction and identification of recombinant Ad-m ALR and Ad-h ALR fixed objective: Recombinant adenovirus the virus containing augmenter of liver regeneration (ALR) gene. Methods: to construct recombinant recombinant shuttle vector (P Ad Track-TO4-m ALR and P Ad Track-TO4-h ALR), recombinant adenovirus (Ad-GFP-m ALR and Ad-GFP-h ALR) was constructed by homologous recombination method, the 4 round of amplification to obtain high low recombinant adenovirus infected L02 cells. And through the expression of fluorescent protein to understand the infection rate, the expression of ALR protein and its proliferation activity was detected by Western Blotting method and CCK-8 method. Results: 1. recombinant adenovirus (Ad-GFP-m ALR and Ad-GFP-h ALR) were successfully constructed recombinant.2. Adenovirus (Ad-GFP-m ALR and Ad-GFP-h ALR) can efficiently and stably expressed in L02 cells infected with.3. and non infection group and empty virus group, overexpression of ALR could promote the proliferation of L02 cells. Conclusion: the successful construction of the recombinant adenovirus Ad-GFP-m ALR and Ad-GFP-h ALR, 15KD ALR has confirmed the experimental cell proliferation effect on the second part: L02 cells. Overexpression of ALR in palmitic acid induced Hep G2 cell fatty degeneration of the role of objective: To observe the expression of 15KD ALR in palmitic acid (palmitic acid PA) Hep G2 induced steatosis, and briefly discusses its mechanism. Methods: the experiment was divided into 5 groups: non infected glands the virus without Hep G2 cell group PA treatment (G2-NC-No infection), Hep G2 cells group PA virus load (G2-Ad-GFP-NC), Hep G2 virus infection load cell group PA (G2-Ad-GFP-PA), Hep G2, Ad-GFP-h ALR cells group without PA infection (G2-A D-GFP-h ALR-NC, Hep G2) infected cells group Ad-GFP-h ALR PA (G2-Ad-GFP-h ALR-PA). Triglyceride (Triglyceride, TG) enzyme and Nile red staining method of qualitative and quantitative detection of intracellular lipid droplets accumulation, Real Time-PCR and Western blotting method was applied to the intracellular solid alcohol regulation element binding protein -1 (SREBP-1), adipose differentiation related protein (ADRP) and peroxisome proliferator activated receptor alpha (PPAR- alpha) detection, the rate of apoptosis was detected by flow cytometry, detection of MAPK pathway related protein Western Blotting method. Results: 1. G2-NC-No and the control group, infection, G2-Ad-GFP-NC, G2-Ad-GFP-h ALR-NC, G2-Ad-GFP-PA TG of the experiment group is obvious the increase of red fluorescence intensity of lipid droplets is significantly enhanced. Compared with G2-Ad-GFP-PA group, expression of ALR (G2-Ad-GFP-h ALR-PA group), intracellular lipid droplets and TG red fluorescence intensity obviously The down-regulation of.2. and control group G2-NC-No, infection, G2-Ad-GFP-NC, G2-Ad-GFP-h compared to ALR-NC, SREBP-1, G2-Ad-GFP-PA in the experimental group in PPAR- RNA and protein expression of M alpha, ADRP were significantly increased. Compared with G2-Ad-GFP-PA group, expression of ALR (G2-Ad-GFP-h ALR-PA group), intracellular SREBP-1, PPAR- alpha, m RNA and ADRP protein expression compared with the experimental group and the control group G2-NC-No.3. G2-Ad-GFP-PA by infection, G2-Ad-GFP-NC, G2-Ad-GFP-h ALR-NC, G2-Ad-GFP-PA in the experimental group the apoptosis rate was significantly increased. But compared with G2-Ad-GFP-PA group, expression of ALR (G2-Ad-GFP-h ALR-PA group) after the Hep apoptosis rate of G2 cells significantly decreased the.4. and control group G2-NC-No infection, G2-Ad-GFP-NC G2-Ad-GFP-h, ALR-NC compared with the phosphorylation of protein p-JNK, G2-Ad-GFP-PA in the experimental group in p-p42/44, p-p38 relative expression increased significantly. But compared with G2-Ad-GFP-PA group, expression of ALR (G2-Ad-GFP-PA) In the group of phosphorylated proteins p-JNK, p-p42/44, p-p38 expression was significantly reduced. Conclusion: fatty degeneration of.2. 1. palmitic acid can induce Hep G2 cells. The expression of lipid droplet accumulation of.3. ALR can reduce PA induced Hep G2 cells overexpression of ALR can reduce the key enzyme in fat metabolism in (SREBP-1, PPAR- alpha, ADRP). The expression of.5..4. ALR can reduce the expression of apoptosis induced by PA in Hep G2 cells the overexpression of ALR can inhibit the phosphorylation of MAPK pathway protein (p-JNK, p-p42/p44, p-p38) is produced.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R575

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