發(fā)布時(shí)間：2018-04-08 22:35

  本文選題：肝再生增強(qiáng)因子　切入點(diǎn)：腺病毒　出處：《重慶醫(yī)科大學(xué)》2016年碩士論文

【摘要】：背景:近年來(lái)非酒精性脂肪肝病(Non-alcoholic fatty liver disease,NAFLD)發(fā)病率明顯上升,西方發(fā)達(dá)國(guó)家的年發(fā)病率高達(dá)20%-40%,在中國(guó)發(fā)病僅次于病毒性肝病,且有極大可能發(fā)展至肝硬化、肝癌以及肝功能衰竭,危及生命。報(bào)道顯示,與普通人群相比,脂肪肝患者的心血管疾病死亡率較高。因此,NAFLD是人類健康的一大嚴(yán)重威脅。且NAFLD的臨床表現(xiàn)輕微,無(wú)特異性表現(xiàn),診斷多依賴實(shí)驗(yàn)室檢查,也無(wú)批準(zhǔn)的單藥治療方案,因此對(duì)NAFLD的發(fā)病機(jī)制進(jìn)一步研究以尋找有效的分子標(biāo)志物用于NAFLD的早期篩查,并發(fā)現(xiàn)新的治療靶點(diǎn)予以針對(duì)性治療,這對(duì)提高早期診斷率及改善預(yù)后意義重大。肝再生增強(qiáng)因子(augmenter of liver regeneration,ALR),首次發(fā)現(xiàn)是定位于肝細(xì)胞漿,具有促進(jìn)細(xì)胞增殖、抗細(xì)胞凋亡,調(diào)節(jié)免疫,逆轉(zhuǎn)肝臟纖維化等作用的一種非特異性細(xì)胞因子。ALR在體內(nèi)發(fā)現(xiàn)有三種分子片段:15KD、21KD和23KD。15KD是23KD的C′末端片斷,即15KD ALR無(wú)信號(hào)肽序列,目前研究23KD ALR主要是在線粒體功能活動(dòng)發(fā)揮作用。已有研究表明特異性敲除小鼠23KD的ALR基因后,肝臟的脂肪性肝炎及肝癌的發(fā)病率明顯增加,而且在NAFLD的病人肝組織標(biāo)本檢測(cè)中發(fā)現(xiàn)23KD ALR的表達(dá)較正常人肝是下調(diào)的,說(shuō)明23 KD ALR在NAFLD的發(fā)生發(fā)展中起重要作用,而15KD ALR作為23KDALR的剪切片段,目前國(guó)內(nèi)外研究中暫無(wú)報(bào)道其在NAFLD中的生物學(xué)作用。因此,本實(shí)驗(yàn)主要構(gòu)建重組15KD ALR腺病毒及探討過(guò)表達(dá)ALR對(duì)軟脂酸誘導(dǎo)Hep G2細(xì)胞脂肪變性的作用,為NAFLD的診治提供新的思路。本研究包括兩個(gè)部分:第一部分:重組Ad-m ALR和Ad-h ALR的構(gòu)建和鑒定目的:重組腺病毒含肝再生增強(qiáng)因子(ALR)基因的構(gòu)建。方法:基因重組法構(gòu)建重組穿梭載體(p Ad Track-TO4-m ALR和p Ad Track-TO4-h ALR),重組腺病毒(Ad-GFP-m ALR和Ad-GFP-h ALR)采用同源重組法構(gòu)建,4輪擴(kuò)增后獲得高低度重組腺病毒。感染L02細(xì)胞,通過(guò)熒光蛋白的表達(dá)了解其感染率,ALR蛋白表達(dá)情況和其增殖活性則通過(guò)Western Blotting法和CCK-8法檢測(cè)。結(jié)果:1.重組腺病毒(Ad-GFP-m ALR和Ad-GFP-h ALR)被構(gòu)建成功。2.重組腺病毒(Ad-GFP-m ALR和Ad-GFP-h ALR)能高效感染并穩(wěn)定表達(dá)于L02細(xì)胞。3.與非感染組和空載病毒組相比,過(guò)表達(dá)ALR均能促進(jìn)L02細(xì)胞的增殖。結(jié)論:成功構(gòu)建重組腺病毒Ad-GFP-m ALR和Ad-GFP-h ALR,細(xì)胞實(shí)驗(yàn)證實(shí)15KD ALR對(duì)L02細(xì)胞具有促增殖作用。第二部分:過(guò)表達(dá)ALR在軟脂酸誘導(dǎo)Hep G2細(xì)胞脂肪變性中的作用目的:觀察過(guò)表達(dá)15KD ALR在軟脂酸(palmitic acid,PA)誘導(dǎo)Hep G2細(xì)胞脂肪變性的作用,并簡(jiǎn)要探討其機(jī)制。方法:本實(shí)驗(yàn)分為5組:未感染腺病毒且無(wú)PA處理的Hep G2細(xì)胞組(G2-NC-No infection),感染空載病毒無(wú)PA的Hep G2細(xì)胞組(G2-Ad-GFP-NC),感染空載病毒有PA的Hep G2細(xì)胞組(G2-Ad-GFP-PA),,感染Ad-GFP-h ALR無(wú)PA的Hep G2細(xì)胞組(G2-Ad-GFP-h ALR-NC),感染Ad-GFP-h ALR有PA的Hep G2細(xì)胞組(G2-Ad-GFP-h ALR-PA)。甘油三酯(Triglyceride,TG)酶法和尼羅紅染色法定量和定性檢測(cè)胞內(nèi)脂滴儲(chǔ)積情況,Real Time-PCR和Western blotting法被應(yīng)用于細(xì)胞內(nèi)固醇類調(diào)節(jié)原件結(jié)合蛋白-1(SREBP-1),脂肪分化相關(guān)蛋白(ADRP)和過(guò)氧化物酶體增殖物激活受體-α(PPAR-α)的檢測(cè),流式細(xì)胞儀檢測(cè)凋亡率,Western Blotting法檢測(cè)MAPK通路相關(guān)蛋白。結(jié)果:1.與對(duì)照組G2-NC-No infection、G2-Ad-GFP-NC、G2-Ad-GFP-h ALR-NC相比,實(shí)驗(yàn)組G2-Ad-GFP-PA的TG的測(cè)定是明顯增加的,脂滴的紅色熒光強(qiáng)度是顯著增強(qiáng)的。而與G2-Ad-GFP-PA組相比,過(guò)表達(dá)ALR(G2-Ad-GFP-h ALR-PA組)后,細(xì)胞內(nèi)的TG及脂滴的紅色熒光強(qiáng)度明顯下調(diào)。2.與對(duì)照組G2-NC-No infection、G2-Ad-GFP-NC、G2-Ad-GFP-h ALR-NC相比,實(shí)驗(yàn)組G2-Ad-GFP-PA中SREBP-1、PPAR-α,ADRP的m RNA及蛋白的表達(dá)均顯著增加。與G2-Ad-GFP-PA組相比,過(guò)表達(dá)ALR(G2-Ad-GFP-h ALR-PA組)后,胞內(nèi)SREBP-1、PPAR-α,ADRP的m RNA及蛋白表達(dá)較實(shí)驗(yàn)組G2-Ad-GFP-PA下調(diào)。3.與對(duì)照組G2-NC-No infection、G2-Ad-GFP-NC、G2-Ad-GFP-h ALR-NC相比,實(shí)驗(yàn)組G2-Ad-GFP-PA中的凋亡率是明顯增加的。但與G2-Ad-GFP-PA組比較,過(guò)表達(dá)ALR(G2-Ad-GFP-h ALR-PA組)后Hep G2細(xì)胞的凋亡率明顯降低。4.與對(duì)照組G2-NC-No infection、G2-Ad-GFP-NC、G2-Ad-GFP-h ALR-NC相比,實(shí)驗(yàn)組G2-Ad-GFP-PA中的磷酸化蛋白p-jnk、p-p42/44、p-p38相對(duì)表達(dá)明顯增加。但與G2-Ad-GFP-PA組比較,過(guò)表達(dá)ALR(G2-Ad-GFP-PA)組中磷酸化蛋白p-jnk、p-p42/44、p-p38相對(duì)表達(dá)明顯下調(diào)。結(jié)論:1.軟脂酸能誘導(dǎo)Hep G2細(xì)胞的脂肪變性。2.過(guò)表達(dá)ALR可減輕PA誘導(dǎo)Hep G2細(xì)胞的脂滴儲(chǔ)積。3.過(guò)表達(dá)ALR可降低脂肪合成代謝中關(guān)鍵酶(SREBP-1、PPAR-α、ADRP)的表達(dá).4.過(guò)表達(dá)ALR可減少PA誘導(dǎo)Hep G2細(xì)胞的凋亡的發(fā)生。5.過(guò)表達(dá)ALR可抑制MAPK通路中磷酸化蛋白(p-JNK,p-p42/p44,p-p38)的產(chǎn)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.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R575

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