Eukaryotic Expression Of Recombinant Human Hepatocyte Growth Factor(rhHGF)and The Therapy Effect Of RhHGF On Experimental Murine Liver Fibrosis

Objective Hepatoeyte growth factor(HGF) was originally identified in partially hepatectomized rats and in rat platelets as the most potent stimulator of DNA systhesis in primarily cultured hepatocyte. Further studies show that HGF is a mesenchymally derived heparin-binding glycoprotein and involves in the regulation of growth, motility, and morphogenesis of various types of cells. HGF is also considered to protect liver from injury and improve hepatic function. This dissertation aims at: (1) expression of recombinant human HGF (rhHGF) in CHO-Kl cells and partially purification of rhHGF; (2) Since the expression of rhHGF by recombinant CHO-K1 cells was at low level, it is ha~d to prepare large scale of rhHGF for in vivo experiments. For this reason, an eukaryotic transient expression vector-pUC SR a IrhHGF- was administrated to mice for assessment of the therapy effect of rhHGF on CC14 induced murine liver fibrosis. Methods and results The study is devided into 3 parts according to individual purpose. 1. Expression of rhHGF pEE14/rhHGF eukaryotic expression vector was extracted from E. coli by alkaline lysis, purified by PEG pricipitation, and tranfected into CHO-kl cells with the help of lipofectin reagent. Positive cell clones were selected out under the pressure of methionine sulfoxmine(MSX). A 396 bp fragment, which is specific to human HGF mRNA, could be amplified from positive cells by reverse transcription polymerase chain reaction(RT-PCR), suggesting the expression of rhHGF in mRNA level. Enzyme linked imniuno sorbance assay (ELISA) showed that the -6- concentration of rhHGF in the supematant of positive cells was around 8---- 10 i-~gIL, suggesting the expression of rhHGF in protein level. By measuring the [3H]-thymidine incorporation into cells, the culture supernatant of positive CHO-K1 cells was found to induce DNA synthesis of primarily cultured rat hepatocytes, suggesting that rhl-IGF was bioactive. 2. Partially purification of rhHGF Components with mollecular weight below 1 OKD was removed from culture supematant by ultrafiltration. The remnant ~vas applied to a heparin-sepharose CL6B affinity column attached to a FPLC apparatus. Unbound material was washed out with 0.01 M Tris-Cl buffer (pH7.4)and the bound material .was eluted with a linear gradient of 0? 2.0 M NaC1 at a flow rate of lmL/min. Fractions of 10 mL were collected and subjected to ELISA for the presence of rh}{GF. The results showed that rhHGF was mainly washed out between 0.9---- 1 .3M NaC1 concentration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that this partially purified rhHGF presented as one band with a MW of 6OKD under non-reducing cinditions. Under reducing conditions it separated into two bands with MW of 62 and 34 KD, respectively. This partially purified rhHGF induced greatly the DNA synthesis of rat primarily cultured hepotocytes. 3.HGF gene therapy of murine liver fibrosis induced by CC!4 Since the concentration of rhHGF in the supematant of positive CHO-K1 cells was about 8 10 IWL, it is laboursome to prepare large scale of rhHGF for in vivo experiments. For this reason, an eukaryotic transient expression vector-pUC SR a /rhHGF-was employed to assess the therapeutic effect of rhHGF on murine liver fibrosis induced by CC14. Mice were injected subcutaneously with Cd4 for 8 weeks to produce liver fibrosis model. 50 pg pUC SR...