Construction Of Recombinant Adenovirus Carring Augmenter Of Liver Regeneration And The Effect Of ALR In Palmitic Acid-induced Lipid Accumulation In HepG2 Cells

Background: The prevalence of non-alcoholic fatty liver disease(NAFLD) is increasing significantly in recent. For the western developed countries,the incidene ranges from 20% to 40% and for China it’s second only to the viral liver diseases. NAFLD own great risk progressing to cirrhosis, liver cancer and liver failure. Comparing with the general population, higher cardiovascular mortality occurs in NAFLD patients. So NAFLD is a serious threat to human health. In addition, the manefestaions of NAFLD are mild and non-specific. The diagnosis of NAFLD mostly depends on the laboratory and imaging examination and no single drug has been approved for NAFLD. Therefore, the pathogenesis of NAFLD needs to be explored further to search for effective molecular marker for early NAFLD screening and new therapeutic targets to have improvement on the early diagnosis rate and the prognosis.Augmenter of liver regeneration(ALR), which located in cytoplasm, is a non-specific cytokine. It can promote cell proliferation, ameliorate cell apoptosis and liver fibrosis and regulate immune. There are three kinds of molecular fragment found in vivo: 15 KD, 21 KD and 23 KD, and the former is C’ terminal fragment of the latter. 15 KD ALR has no signal peptide sequence and 23 KD ALR is mainly involved in mitochondrial function activities. It has been reported that the incidence of steatohepatitis and hepatocellular carcinoma was significantly increased in the mice with specific 23 KD ALR gene knock down and the expression of ALR protein was reduced in patients with NAFLD comparing with those in normal people. However, there are no report on the function of 15 KD ALR in the pathogenesis of NAFLD. Therefore, the aims of the study are to construct the recombinant adenovirus ALR, explore the role of overexpression of ALR in HepG2 cells with palmitic acid(PA) treatment and provide new ideas for the diagnosis and therapy of NAFLD.This study contains two parts:Part1: Construction and identification of recombinant adenovirus mouse ALR and human ALR(Augmenter of Liver Regeneration)Objective: To construct recombinant adenovirus carrying ALR gene.Methods: recombinant DNA technology and homologous recombination method were utilized to construct recombinant shuttle vector(pAdTrack-TO4-mALR and pAdTrack-TO4-h ALR) and recombinant adenovirus(Ad-GFP-mALR and Ad-GFP-hALR) respectively. After four cycles of amplification, high titers of the recombinant adenovirus Ad-GFP-mALR and Ad-GFP-hALR were obtained. We evaluated the recombinant adenovirus infection efficiency by the expression of green fluorescent protein, assessed the expression of ALR protein by western blotting and detected the proliferation activity of L02 cells when L02 cells were infected with either Ad-GFP-mALR or Ad-GFP-hALR by CCK-8 assay.Results:1. pAd Track-TO4-mALR, pAdTrack-TO4-hALR, Ad-GFP-mALR andAd-GFP-hALR were constructed successfully.2. Both of Ad-GFP-mALR and Ad-GFP-hALR could infect HepG2cells efficiently and expressed steadily.3. The HepG2 cells with the infection of either Ad-GFP-mALR orAd-GFP-hALR had significantly proliferation activity comparingwith those uninfected and those infected with Ad-GFP.Conclusion: We constructed recombinant adenovirus Ad-GFP-mALR and Ad-GFP-hALR successfully and the over expression of either mALR and hALR can promote the proliferation activity of L02 cellsPart 2: The effect of over expression of ALR on palmitic acid-induced lipid accumulation in Hep G2 cellsObjective: To observe the effect of over expression of ALR in Hep G2 cells treated with palmitic acid(PA) and explore the mechanism.Methods: Five groups in the experiment: Hep G2 cells without adenovirus and PA treatment(G2-NC-No infection), Hep G2 cells treated with Ad-GFP instead of PA(G2-Ad-GFP-NC), Hep G2 cells with both of Ad-GFP and PA treatment(G2-Ad-GFP-PA), Hep G2 cells treated with Ad-GFP-h ALR instead of PA(G2-Ad-GFP-h ALR-NC), Hep G2 cells with both of Ad-GFP-h ALR and PA treatment(G2-Ad-GFP-h ALR-PA). Triglyceride(TG) enzymatic method and Nile red staning were applied to detect the content of intracellular lipid droplets qualitatively and quantitatively. Real-time PCR and western blotting were used to evaluate the expression of sterol regulatory element binding protein-1(SREBP-1), peroxisome proliferator activated receptor-α(PPAR-α), adipose differentiation related protein(ADRP). Flow cytometry was employed to assess the apoptosis rate and western blotting was used to detect the expression the MAPK pathways.Results:1. Comparing with the control groups G2-NC-No infection, G2-Ad-GFP-NC and G2-Ad-GFP-h ALR-NC, the content of TG and the intensity of red fluorescence in group G2-Ad-GFP-PA were significantly enhanced. Comparing with G2-Ad-GFP-PA, over expression of ALR(G2-Ad-GFP-h ALR-PA) lowered the content of TG and the intensity of red fluorescence significantly.2. Comparing with the control groups G2-NC-No infection, G2-Ad-GFP-NC and G2-Ad-GFP-h ALR-NC, the expression of m RNA and protein of SREBP-1, PPAR-α and ADRP in G2-Ad-GFP-PA group were significantly up-regulated. Comparing with G2-Ad-GFP-PA, over expression of ALR(G2-Ad-GFP-h ALR-PA) decreased significantly the expression of m RNA and protein of SREBP-1, PPAR-αand ADRP.3. Comparing with the control groups G2-NC-No infection, G2-Ad-GFP-NC and G2-Ad-GFP-h ALR-NC, the apoptosis rate in G2-Ad-GFP-PA was significantly elevated. After over expression of ALR, the apoptosis rate was reduced statistically.4. Comparing with the control groups G2-NC-No infection, G2-Ad-GFP-NC and G2-Ad-GFP-h ALR-NC, expression of phosphorylated protein(p-jnk、p-p42/p44、p-p38) were significantly increased in the group G2-Ad-GFP-PA. However, over expression of ALR(G2-Ad-GFP-h ALR-PA) down-regulated significantly the phosphorylated protein expression.Conclusion:1. PA could lead to the lipid accumulation in Hep G2 cells.2. Over expression of ALR could lower the lipid accumulation of Hep G2 cells treated with PA.3. Over expression of ALR could down-regulate the key enzymes in lipogenesis and lipid metabolism(SREBP-1、PPAR-α、ADRP).4. Over expression of ALR reduced the apoptosis rate of Hep G2 cells induced by PA.5. Over expression of ALR restrained the expression of phosphorylation of MAPK pathways-associated protein(p-JNK,p-p42/p44,p-p38)...