Expression Of HSA-IGF-1 And HSA-eTGFBR2 In Chinese Hamster Ovary Cells And Analysis Of Anti-liver Fibrosis Activity

Insulin like growth factor(IGF-1),mainly synthesized and secreted by the liver,is closely related to liver function.It plays central roles in reducing liver oxidative damage,reducing liver fibrosis and improving liver function.Transforming growth factor ?1(TGF-?1)is the strongest liver fibrosis-inducing factor.It can inhibit hepatocyte growth and activate hepatic stellate cells(HSC)via TGF-?1/Smads pathway,which suggests that TGF-?1 can be treated as a therapeutic target for liver fibrosis treatment.The extracellular domain of TGF-?1 receptor type ?(eTGFBR2),binding partner for TGF-?1,can antagonize the activity of TGF-?1 and play an important role in the treatment of hepatic fibrosis.Small molecule protein drugs(< 20 kD)are easy to be glomerular filtration or protease degradation in vivo and have short half-lives,resulting in high dose and high frequency injection to obtain a certain therapeutic effect on clinical utility.In order to improve the stability of small molecule proteins,human serum albumin(HSA)fusion technology was used to construct HSA-IGF-1 and HSA-eTGFBR2 fusion protein,which were stable and high expressed in Chinese hamster ovary(CHO)cells.After a series of purification,the bioactive long-term fusion proteins were obtained.Then,the anti-liver fibrosis activity of these two fusion proteins were evaluated in vitro and in vivo,including the function of inhibiting liver fibrosis and improving liver function.The main results are shown as follows:(1)The hsa-igf-1 fusion fragment was gained by HSA fusion technology and inserted into eukaryotic expression vector pMH3.The recombinant expression vector was transfected in CHO cells and stable and high expressed cell clones were obtained by G418 pressure selection and monoclonal selection.The highest expression cell clone was was cultured in fed-batch suspension culture for 8 days.The highest viable cell density reached 8.4×106 cells·mL-1 and the final yield of HSA-IGF-1 was 100 mg·L-1.The fusion protein was then purified using a three-step chromatographic procedure,including Blue,Octyl and Q column,resulting in about 94% purity and 31.5% recovery rate.Compared with IGF-1,the purified HSA-IGF-1 had similar biological activities and could stimulate NIH3T3 cells proliferation via PI3K/AKT signaling pathway.(2)The hsa-etgfbr2 fusion fragment was gained by HSA fusion technology and recombinant expression vector was constructed and transfected in CHO cells.After G418 pressure selection and monoclonal selection,stable and high expressed cell clones were obtained and cultured in fed-batch suspension culture for 8 days.The highest viable cell density reached 8.5×106 cells·mL-1 and the final yield of HSA-eTGFBR2 was 180 mg·L-1.Using a three-step chromatographic procedure,that is Blue,Octyl and Q column,HSA-eTGFBR2 with a purity about 91% was obtained and the recovery rate of whole purification process was 36.8%.HSA-eTGFBR2 could bind to TGF-?1 with a KD value 1.42×10-8 mol·L-1.Moreover,HSA-eTGFBR2 could antagonize the inhibitory effects of TGF-?1 on hepatocyte cells.(3)The anti-liver fibrosis activities of HSA-IGF-1 and HSA-eTGFBR2 were detected in vitro.HSA-IGF-1 could reverse the growth inhibition effects of TGF-?1 and protect the L-02 cells from injury.HSA-eTGFBR2 could bind TGF-?1 and antagonize its proliferation effects on HSC.The activation of hepatic stellate cell line(HSC-T6)induced by TGF-?1 were used as a liver fibrosis model in vitro.HSA-IGF-1 and HSA-eTGFBR2 could reduce the expression of ?-SMA,COL?,TIMP-1 and the supernatant level of COL? COL?,increase the level of MMP-2,resulting in inhibiting the activation of HSC-T6 and ECM accumulation.Then the mechanism was preliminary discussed.These two fusion proteins could interfere the TGF-?1/Smads signaling pathway by inhibiting the phosphorylation of Smad3,resulting in inhibiting TGF-?1 bioactivity and liver fibrosis.(4)Analysis anti-liver fibrosis activities of HSA-IGF-1 and HSA-eTGFBR2.CCl4 could cause liver structure disorder,hepatocellular necrosis,collagen fibers proliferation,leading to liver fibrosis.Hepatoprotective drug silymarin could significantly improve liver structure.But albumin had little effect on liver fibrosis treatment.HSA-IGF-1 and HSA-eTGFBR2 could significantly improve the symptoms of liver fibrosis,reduce the damage of liver cells and collagen deposition,and recovery the liver basic structure to normal.Also,they could improve liver function and reduce the expression level of liver fibrosis marker ?-SMA and COL?.The intervention group was better than the treatment group,suggesting that early treatment is better in liver fibrosis.Moreover,reducing the injection frequency of HSA-IGF-1 and HSA-eTGFBR2 could also achieve good effects,which suggested that these two fusion proteins had good anti-liver fibrosis activities.