Study On Fermenter Preparation Technology And Related Anti-liver Fibrosis Bioactivity Of Recombinant Human Kallistatin

In recent years, a growing number of studies have shown that Kallistatin（KAL）, anendogenous angiogenesis inhibitor, not only has roles of anti-angiogenesis andvasodilatation, but also exerts significantly multiple functions such as antioxidant,anti-inflammatory and anti-tumor.Our laboratory studies showed that KAL has a significantly therapeutic effect onCCl4caused mice liver fibrosis through a speculated multi-channel regulation onoxidative stress, inflammation, angiogenesis and portal hypertension in the fibroticliver. This thesis, based on the in vivo study, explored the related anti-fibrosisactivities of KAL through a variety of in vitro models.For preparation of biologically active target protein, the thesis first to explore thefermenter technology of KAL through filtration of genetic engineering pichia pastoristhat high and efficient expression of recombinant human KAL protein. High purity ofKAL protein was achieved by fermentation and further isolation, purification,identification with Western blot and Elisa quantitative analysis. The purity ofrecombinant human KAL was over95%eventually by analysis of gel imaging system.The highest KAL concentration of the fermentation supernatant is up to130mg/L.After purification, KAL protein at molecular weight of40kD was obtained.Next, in vitro bioactivities were studied as follows:①Three oxidative stressmodels （H₂O₂, tBHP, Fe-NTA） are constructed in three liver fibrosis related celllines （Changliver, HUVEC, Fe-NTA）to investigate the antioxidant capacity ofrecombinant human KAL protein.②The role of KAL protein in the migration andtube formation of HUVEC was investigated. And the effect of KAL on primaryculture activated rat hepatic stellate cell was studied.Experimental results show that:①KAL can significantlly protct cell injury resultfrom H2O2-induced oxidative stress, reduce intracellular superoxide radical （O₂-）and protect cell from H2O2-induced apoptosis in three cell lines.②KAL caninhibite the migration and tubule formation of vascular cells in a dose-dependent way, and the killing capacity on culture activated primary rat hepatic stellate cells is moresignificantly than LX-2.This study provides a better understanding of KAL function, and lays a foundationfor its further study and clinic application in liver fibrosis.