The Effect Of Recombinant Human Kallistatin In Hepatic Sinusoidal Endothelial Cells

Kallistatin(Kal)exerts pleiotropic activates in inhibiting inflammation,apoptosis,and repair of fibrosis.Hepatic sinusoidal endothelial cells(HSEC)occupies the largest number in liver nonparenchymal cells.The transformation of the phenotype and physiological characteristics will lead to the changes in the microcirculation of liver,causing capillarity of hepatic sinusoid,and it is an important pathological change of liver fibrosis.Hepatic sinusoidal endothelial cells have lots of fenestrae and lack basement membranes.Not only can VEGF stimulate liver cells or hepatic stellate cells to produce paracrine but stimulate eNOS of HSEC to secrete NO to maintain the fenestrae.This thesis is to explore the role of Kal in liver sinusoidal endothelial cells and its anti-fibrosis function by adopting in vitro models.This thesis adopts the Pichia pastoris containing plasmid pPIC9-Kal/GS115,which is preserved in our laboratory,to ferment in a high density.After separating and purifying,we will botain rhKal in the end.The purity of purified rhKal can reach more than 95% by means of Western Blot,Elisa and gel imaging system.Each time we can get 70 mg rhKal averagely,and its molecular mass is about 40 kd.By adopting collagenase in situ perfusion,Percoll density gradient centrifugation and selective adherent the rat liver sinusoidal endothelial cells were separated.While experimenting,optical microscope and scanning electron microscope were used to explore the morphological character and ultrastructure of HSEC.Additionally,CD31,CD34,CD14,CD45 were used to identify HSEC in immunohistochemical staining.In this article,by isolating and culturing(1.78±0.33)×107 HSEC was abtained within one rat,and its activity is more than or equal to 95%,the purity reached to 92%.Under light microscope,we can find typical cells,and fenestrae can be observed under SEM.CD31,CD34,CD14 staining were positive,while CD45 staining was negative in HSECs.In vitro experiments include the fuction of rhKal in the maintenance of HSEC fenestrae,the influence on eNOS activity,NO level and gene expressions.The relationship between the function of rhKal and the PI3K-AKT-eNOS pathway was also observed in this article.The experimental results show that Kallistatin can maintain the fenestrae of HSECs,the role of maintaining can be inhibited by LY,L-NAME.Kallistatin can also make the activity of NOS increase 1.4 times(n=3,P<0.05);the expression and the level of NOx can be promoted by Kallistatin to 1.7 times(n=3,P<0.05).The promoting effect of Kallistatin on eNOS phosphorylation and NO secretion can be inhibited by the PI3 K inhibitor LY,which shows that the activation of Kallistatin mediated secretion of eNOS and NO is completed through the PI3K-Akt pathway.This article provides a new direction and theoretical basis for the anti fibrosisprinciple of Kallistatin.