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Objective To study the relationship between CXXC motif and biologic activity of human augmenter of liver regeneration protein(hALRp).Methods The function of CXXC motif and its relationship with bioactivity were investigated between hALRp and hALRp-C65A groups by detecting the sulfhydryl oxidase activity;the interaction of target protein with GST-Na~+,K~+-ATPase fusion protein was examined by GST-Pulldown and MTT was used to study the ability of hALRp-C65A to promote HL-7702 hepatic cells proliferation in vitro.Results The turnover number(TN) of hALRp in sulfhydryl oxidase activity assay was 1.25?0.21,while TN was 0 of hALRp-C65A,there was significant difference between them(P
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Objective:To detect the existence of expression of human augmenter of liver regeneration(hALR) in HepG2 cells,and to observe proliferation of HepG2 cells after neutralizing hALR with anti-hALR McAb.Methods:the expression of hALR in HepG2 cells was observed immunocytochemistry and reverse transcriptive PCR(RT-PCR).Proliferation of HepG2 cells after neutralization was detected by ~3?H-TdR incorporation approach.Results:① hALR was expressed by HepG2 cells.② Anti-hALR McAb inhibited partially the autonomous growth of HepG2 cells.Conclusion:Anti-hALR McAb inhibit the autonomous growth of hepatoma cells partially;moreover,hALR maintain the autonomous growth of hepatoma cells in vitro through autocrine mechanism.
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Objective To investigate the influence of recombinant human augmenter of liver rege neration (hALR) on stromelysin 1 gene expression in experimental liver fibrosis. Methods Two kinds of rat model of experimental liver fibrosis induced by CCl 4 and albumin were established and different dosages of recombinant human augmenter of liver regeneration were given during the process of model making. Total RNA of liver tissues was extracted and stromelysin 1 gene expression levels were measured by reverse transcription polymerase chain reaction (RT PCR). Results In both rat models of experimental liver fibrosis, stromelysin 1 gene expression levels in hALR treating group were significantly higher than those of model groups in different periods of model forming. Stromelysin 1 gene expression levels in high dose hALR treated group were significantly higher than those of low dose hALR treated group.Conclusion Recombinant human augmenter of liver regeneration may have effects of promoting gene expression of stromelysin 1 in experimental liver fibrosis.
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Hepatic stellate cell (HSC-T) was cultured in medium containing different concentrations of recombinant human augmenter of liver regeneration (hALR),and cells were collected at different incubation period. Total RNA of HSC was isolated and collagenⅠ and Ⅲ gene expression levels were measured with reverse transcription polymerase chain reaction. The results showed that, collagen Ⅰ gene expression levels of HSC in high(0 2ng/L),middle (0 02ng/L) and low (0 002ng/L) concentrations of three hALR groups were much lower than those of control group after exposure to hALR at 8h, 24h, 48h and 72h. Collagen Ⅰ gene expression levels of HSC in high dose group were also significantly lower than those of middle and low dose groups . Collagen Ⅲ gene expression levels of HSC in middle and low dose hALR group were much lower than those of control group at 24h,48h and 72h, Collagen Ⅲ gene expression levels in high dose group were significantly lower than those of control group at 8h, 24h, 48h and 72h, and were also much lower than those of middle and low dose groups. It suggested that hALR could inhibit collagen Ⅰ and Ⅲ gene expression in hepatic stellate cells.
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To investigate the influence of recombinant human augmenter of liver regeneration (hALR) on stromelysin 1 gene expression in rats with fibrotic liver. CCl 4 or albumin induced liver fibrosis in rats was established, and different dosages of recombinant human augmenter of liver regeneration were given to rats with liver fibrosis.Liver specimens were obtained at different intervals of treatment , total RNA of liver tissues were isolated and stromelysin 1 gene expression was measured by reverse transcription polymerase chain reaction (RT PCR) . The results showed that in both rat models of experimental liver fibrosis , stromelysin 1 gene expression levels were significantly higher in hALR treated rats than those without the treatment at various intervals. Stromelysin 1 gene expression levels in high dose hALR treatment group were significantly higher than that in low dose hALR treatment group. It suggested that recombinant human augmenter of liver regeneration may enhance stromelysin 1 gene expression in rats with fibrotic liver.
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Objective:Identification of the antigenic determinants of hALR.Methods:Theoretic antigenic determinants of hALR was predicted by using Hopp&Wodds method and and Goldkey software package.The four polypeptides according to the amino acid sequences of the predictive linear epitopes of hALR were synthesized and were used to analyze the antigenic determinants of hALR recognized by antibodies.Results:The polypeptides corresponding to residues 6～5,68～80 and 105～112 of hALR were its antigenic determinants.Conclusion:Prediction of the protein antigenic determinants by computer program and detection of them by competitive ELISA with synthesized polypeptides is a useful method of identification of the antigenic determinants.
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Objective To investigate the influence of recombinant human augmenter of liver regeneration (hALR) on gelatinase A(MMP 2) gene expression in experimental liver fibrosis. Methods Two kinds of animal model of rat experimental liver fibrosis induced by CCl 4 and albumin were established and different dosage of hALR (10,50 ?g?kg -1 ?d -1 )was given during the process of model making. Total RNA of liver tissues was isolated and MMP 2 gene expression levels were measured by reverse transcription polymerase chain reaction (RT PCR). Results In both rat models of experimental liver fibrosis, MMP 2 gene expression levels in two hALR preventing group were significantly lower than those of model group in different periods of model forming. MMP 2 gene expression levels in high dose of hALR treating group were significanfly lower than those of low dose hALR treating group. Conclusion hALR may have an effect on inhibiting gene expression of MMP 2 in experimental liver fibrosis.
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Objective:To construct a recombinant prokaryotic expression vector pQE30-hALRIP for further research.Method:PCR was used to amplify hALRIP codon region.The gene was inserted into pMD18-T vector and sequenced.Then the prokaryotic expression vector pQE30-hALRIP was constructed and identified. Results:Clonal recombinant vector pMD18- hALRIP and expression vector pQE30-hALRIP were constructed. DNA sequence analysis and restriction analysis showed both of the vectors were the same as the predicted results. Conclusion:Prokaryotic expression vector pQE30-hALRIP is successfully constructed.
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In order to investigate the influence of recombinant human liver regeneration augmenter (hALR) on serum hyaluronic acid content in the process of experimental liver fibrosis, two kinds of animal models of rat experimental liver fibrosis induced by CCl 4 and albumin were established and different dosages of hALR were given during establishing the model. Serum specimens were obtained in different period of model establishment, and hyaluronic acid content was measured by radioimmunoassay (RIA). Results showed that in both rat models of experimental liver fibrosis, serum hyaluronic acid content in two hALR treatment group was significantly lower than that in different periods of model establishment. Serum concentration of hyaluronic acid in large dose of hALR treatment group was also significantly lower than that in low dose of hALR treatment group. These results indicate that recombinant human liver regeneration augmenter may decrease serum hyaluronic acid content in experimental liver fibrosis.