| Objective:The incidence of Non-alcoholic fatty liver (nonalcoholic fatty liver disease, NAFLD) increased year by year, and the disease can progress to liver fibrosis and cirrhosis, but its precise mechanism is currently unclear. Our early experiment showed that Microtus fortis is prone to fatty liver, therefore, the purpose of this study is to establish a Non-alcoholic fatty liver model of Microtus fortis, and to study the pathogenesis of Non-alcoholic fatty liver disease in the model of Microtus fortis.Methods:We selected 105 male Microtus fortis in Dongting Lake which are 6-week-old, and randomly divided them into three groups.The three groups were fed the high-fat ingredients (plus 5% lard,0.5% cholesterol and 0.1% bile salt into based on the standard diet for rats and mice, model 1 group), the standard diet (model 2 group), the high-fiber material (the final concentration of Bold fiber was 10%, crude fat was 3% on the basis of the standard diet for rats and mice, control group) respectively.7 voles of each group were sacrificed at the beginning of the study time of 2,4,6,8,12week. During the process of our study we had observed the general situation and changes in liver index of Microtus fortis. ALT, AST, TG, TC, ALP, FFA, r-GT, HDL, LDL, CHE, GLU values were measured by biochemistry analyzer. The histological changes of all voles'livers were observed using light microscopy. Genes which should be possible related to non-alcoholic fatty liver were selectecd. The cDNA-specific primers were designed in the conserved region according to the sequences of mice and rats from the NCBI. Then real-time PCR was done to obtain the gene expression levels of the livers.We selected four Microtus fortis in Dongting Lake which are 12 months of age, after we sacrificed them whichever liver were obtained to extracte total RNA, then The plasmid cDNA library from liver of Microtus fortis was constructed by using SMART technology. The titers and capacity of this cDNA library were determined. Randomly selected a single colony to sequence and calculate the ratio of full-length gene.The plasmid cDNA library of the liver from Microtus fortis was constructed by using SMART technology. The purposed colonies were got through screening libraries by PCR method, and their full-length cDNA sequences were obtained by sequencing with pBluescriptⅡSK universal primers M13R.Results:Voles fed the high-fat diet developed panlobular macrovesicular steatosis, voles fed the standard diet developed light steatosis, whereas those fed the high-fiber diet had normal livers.Compared with the control group, liver weight and liver index of model group 1 were significantly increased (P< 0.05), serum ALT, AST, TC, FFA, r-GT, LDL, CHE and GLU were significantly high (P< 0.05), HDL and TG were significantly low (P < 0.05). Real-time PCR showed that the five genes of CYP2E1, ECHS1, CYP2D5, CYP2a3a and PGC-1 were high-expression (P< 0.05), the three genes of SC4MOL, Adipor and AMPK-1 were low-expression (P< 0.05).Compared with control group, the liver weight and liver index of model group 2 were significantly increased (P< 0.05), the FFA, LDL and CHE also significantly increased (P< 0.05), HDL of blood decreased significantly (P< 0.05), other indexes had no significant difference. Real-time PCR showed that all the gene expression trends are not obvious (P> 0.05).cDNA plasmid library of Microtus fortis liver was constructed by using SMART technology. The titers of this cDNA library were 1076 pfu/ul, the recombination rate was about 94%, the library capacity was 1.08×106, the integrity rate was 77.3%.Three full-length cDNA sequences of Microtus fortis CYP2D5, CYP2E1 and ECHS1 were obtained. The CYP2D5 cDNA is 1865bp in length and contains a 1482bp open reading frame (ORF) encoding a 494 amino acids. The CYP2D5 cDNA is 1690bp in length, and contains a 1514bp ORF encoding 504 amino acids. The ECHS1 cDNA is 1013bp in length, and contains an 873bp ORF encoding 290 amino acids. Sequence analysis reveals that the identity of the three cDNA sequences and deduced amino acids among Microtus fortis, Homo saplens, Mus musculus and Rattus norvegicus is high.We have registerd the three full-length cDNA sequences in the GenBank, registration are respectively GQ507485, GQ507486, GQ845171. |
