The Expression Of A Novel Rat STGFβRⅡ-IFNγ Fusion Protein And Its Inhibitory Effect On Rat Hepatic Fibrosis

Hepatic fibrosis is a pathological process shared by a variety of causes in response to hepatocyte necrosis and inflammatory insults, which is mainly characterized by the excessive deposition of extracellular matrix (ECM) in Disse space in the liver. Activated hepatic stellate cells (HSC) are known to be the primary ECM-producing cells in hepatic fibrogenesis. The activation of HSC is largely mediated by transforming growth factor (TGF) p, which is actually the most potent pro-fibrogenic cytokine as reported. Thus the TGFp/HSC axis is widely considered a potential target for anti-hepatic-fibrosis therapy by a great number of investigators. TGFβ exhibits its biological function via the TGFp receptors (TpR), including type I, II and III receptors. Both the dominant-negative TβII and the soluble TβR II (soluble TGFβRII, sTβRII) can bind to TGFp but lack the intracellular kinase domain which is essential to initiate signal transduction. Other studies have proved that IFNγ can induce the expression of Smad7, inhibit the interaction between TpR complex and Smad3, which indirectly inhibits TGFβ activity and HSC activation, sTβR II and IFN-γ act at different sites to block the activity of TGFβ and theactivation of HSC, so the combined application of them may produce a synergic effect and in turn reduce their required dose to lower the side effect.Cytokine recombinant fusion protein refers to a category of artificial protein products produced by genetically fusing the encoding fragments of cytokines and/or other proteins with specific biological functions. This kind of proteins exerts strongly enhanced biological activities when compared with the original individual proteins.To investigate the potential synergic anti-fibrotic effect of sT|3R II and IFNy and the enhancement of cytokine activity by fused expression as well, we constructed a prokaryotic expression system of rat soluble TGFpRlI/rat IFN-y (RsTpR II -IFNy ) fusion protein and separate, purify this RsTpR II -IFNy fusion protein. We also identify the bioactivities of this recombinant fusion protein which exhibits a combined effect of RsTpR II and RIFN-yin inhibiting the rat hepatic fibrosis, and paves the way for further novel anti-fibrotic drugs studies.Part I. The expression, purification and identification of a novel rat sTGFpRlI -IFNy fusion proteinRsTpRII-IFNy fusion gene was amplified from pSecTag2/ RsTpRlI-IFNy eukaryotic expression vector with specific primers. The pSecTag2/ RsTpR II -IFNy eukaryotic expression vector can express RsTpR II -IFNy fusion protein successfully in the CHO cells. Then RsTpRlI -IFNy fusion fragment was cloned into PET 44 prokaryotic expression vector. The recombinant, having been screened by ampicillin and identified by double digestion and sequencing, was transformed into BL21(DE3) susceptive coliform and express RsTpR II -IFNy fusion protein induced by IPTG. The RsTpR II -IFNy fusion protein was examined by ELISA and western blotting for RIFNy and RsTpRlI expression, respectively. These assays revealed that in thelysate of pET44/RsTpR II-IFN-y transformed BL21(DE3), there is the target protein which can react with RsTpRlI and RIFNy specific antibodies.To investigate the bioactivities of RsTpR II and RIFNy parts of the fusion protein, anti-proliferation-inhibition assay and anti-viral assay were conducted. In our study, the inhibitive effect of TGF|3 on CCL-64 MM epithelial was abrogated by the RsTpRlI-IFNy fusion protein, and this effect is concentration-dependent, which indicated that RsTpR II -IFNy retains RsTpR II bioactivity;in antiviral assay, L929 cells without RsTpR II -IFNy protection quickly exhibits the typical signs of VSV infection, while RsTpRII-IFNy protected L929 turned out to be insensitive to VSV challenge during our observation, and this protective effect is also concentration-dependent, which indicated that RsTpR II -IFNy retains the anti-viral activity of rat IFNy.Conclusion1. We successfully constructed a prokaryotic expression vector of a rat sTpR II -IFNy fusion protein, and obtained a fusion protein with the bioactivities of both moieties.2. Purify this RsTpR II -IFNy fusion protein and the purification do not destroy the biological function of this recombinant fusion proteinPart II. A experimental study of rat sTGFpR II -IFNy fusion protein in therapy of rat hepatic fibrosisTo further investigate the anti-fibrotic effect of this rat RsTpR II -IFNy fusion protein in vivo, we injected this fusion protein into CCL4-induced hepatic fibrosis model rats via subcutaneous administration and observed the potential synergy of the RsTpR II -IFNy fusion protein.A rat persistent hepatic fibrosis model was induced with tetrachloride carbon (CCL4). This induction method is more convenient in operation when compared with bile duct ligation (BDL), and the hepatic fibrosis is more typical. In the pilot study, Rats (n = 130) were subcutaneously administered dissolved CCI4 in vegetable oil (a proportion of 1:1) at 0.3 ml/100 g of body weight, i.p., twice a week for 8 weeks, and then the liver was sent for histopathological observation, which revealed a significant fibrotic change. Another histopathological examination was conducted 5 weeks after the final administration of CCI4, and revealed no significant self recovery in the liver. This method of inducing hepatic fibrosis was applied to the following studies.After establishing the hepatic fibrosis model, we divided the rats randomly into several groups, which were later given RsTpRlI-IFNy fusion protein, RIFNy, RsT(3RII protein, mixture of RIFNy and RsTpRlI protein respectively, every two days for 8weeks Untreated hepatic fibrosis group and normal rats were used as controls. The resolution of hepatic fibrosis was expected through in vivo blocking the activity of TGFp and the activation of HSC.Then six rats were dissected and their livers were observed respectively in 5,6,7and 8 weeks after the first treatment of fusion protein. The pathological fibrosis scores were found to be lower in rats treated with RsTpRH-IFNy fusion protein compared with other groups. All rats were killed two weeks after the end of the treatment and histological sections of their livers were evaluated by hematoxylin and eosin and Sirius red stains. In the mean time, collagen III (Col III), a -smooth muscle actin (a-SMA), TGFpl and TGFPRII were examined with immunohistochemical technique, western blotting technique and real- time RT-PCR technique. We found the content of these four substances and the expression level of their mRNA in the RsTpR II -IFNy treated group were markedly less than those of other groups.Histopathological evidences furthered confirmed that the rat hepatic fibrosis treated with RsT(3R II-IFNy fusion protein was significantly reversed when compared with other groups. Taking the results, we can conclude that fused expression of RsTpR II and RJFNy revealed a combined anti-fibrotic effect both in vitro and in vivo.Conclusion1. We successfully established a persistent rat hepatic fibrosis model with CCL4;2. Therapy the hepatic fibrosis model rats by injection the RsTpR II -IFNy fusion protein subcutaneously can significantly inhibit rat HSC activation induced by CCL4, reducing its TGFpl, TGFpRlI, a-SMA and ECM expression and reversing the hepatic fibrosis. This effect is more potent than those of individual RsTpRlI or RIFNy protein. All the evidences above indicated that fused expression of RsTpR II and RIFNy may exhibits a combined anti-fibrotic effect.