Studies On The Production Of RhKD/APP And Protective Effect Of Hepatic Injury

Serine protease are a group of enzymes that exist in biological body andkeep an equilibrium between serine proteases and their specific inhibitors.Theserine proteases inhibitors play a important role in modulating physiologicalprocess ,such as coagulation, fibrinolysis, inflammatory respone,cell migration,cell differentiation, complement activation and release of hormone of peptideand protein. The bovine pancreatic typsin inhibitor is a representative inhibitorof Kunitz family.BPTI extracted from bovine pancreas is serine proteaseinhibitor which has 58 amino acids with three disulfide bridges.It inhibitstrypsin with 1:1 stoichiometry.BPTI is a nonspecific and broad-spectrum serineprotease inhibitor which affects known serine proteases such astrypsin,chymotrypsin,plasmin and kallikrein. It can defend blood plateletagainst bleeding, treat the patient with cruor impediment, in which situation thehemorrhage may occur. BPTI can be used in cardiac surgery as beingextracorporal circulated to decrease the incidence of bleeding and theinflammatory reaction caused by the surgery. But infusion of high doses ofthese nonmammalian inhibitor could result in immunologic reactions inpatients undergoing repeat use.So we need find a substitute which ispreferable ,small ,potent and of human sequence origin.The amyloid β-proteinprecursor contains a domain homologous to Kunitz-type serine proteaseinhibitors.The sequence of hKD/APP which is consisted of 57 amino acids has43% sequence identity with that of BPTI. The tertiary fold ,reactive center andmechanism of function is very similar to that of BPTI.The isoforms of APPcontaining KD and hKD/APP show ability to inhibit a variety of serineproteases such as factor XIa,epidermal growth factor(EGF)-binding protein,theγ-subunit of nerve growth factor,expecially the inhibition constant (Ki)ofhKD/APP to factor XIa is higher than that of BPTI. The hKD/APP has activityof growth factor and is an effective cell adhesion molecule.They probably playa role in the complex series of events that lead to tissue repair at vascularwound sites and protect neurocytes against injury.But the pathological andphysiological role of this protease inhibitor domain which concerns withabnomal metabolism of APP are not well understood.So we need to produce alarge quantity of rhKD/APP to discover and study the possible mechanisms onpharmacodynamics.In order to improve these situations, studies were done as follows:① we constuct expression vector pPICZα-KD200 and transform it intoPichia pastoris via electroporation. The rhKD/APP secreted into the culturesupernatant with high level by the Pichia pastoris was screened. ② TherhKD/APP was expressed in 80 L fermentor with optimizing parameters onlarge scale and the broth was harvested and purified with cation exchangechromatography and hydrophobic chromatography. ③ Observation of theeffect of rhKD/APP on primarily cultured hepatocytes at the cell level.④Observation of protective effect of rhKD/APP on acute hepatic injury andexperimental chronic liver injury induced by carbon tetrachloride on rats invivo.1 Construction and screening of Pichia pastoris expression system forrhKD/APP.The rhKD/APP expression vector pPICZα-KD200 was constructed byperforming PCR with specific primer with Xho I and Xba I target sites and a partof α-mating factor signal peptite.After identification by endonuclease digestationassay and sequencing , linearized expression vector pPICZα-KD200 wastransformed into Pichia pastoris via electroporation. The transformed Pichiapastoris were cultured in medium with zeocin resistant for 28oC,36 h .Thetransformed Pichia pastoris were screened and the genomic DNA of the screenedyeasts were extracted using PCR technique. Positive yeast clones tested by PCRwas proliferated and then the expression of rhKD/APP was induced with 0.5%methanol. The supernatant of fermentation is identified by SDS-PAGE, inhibitionactivity to trypsin and purified by cation exchenge and hydrophobicchromatography. The results showed that rhKD/APP was identical with the nativehKD/APP in molecular and physics and chemical properties with specific activity8.4 EPU?mg-1.2 Studies on fermentation technology and a new method of purifying rhKD/APPon large-scale:Pichia pastoris has many advantages as a kind of expression host, and it'svery suitable for large-scale expression of the extraneous proteins. So afteridentifying the bioactivities of the rhKD/APP, we explored the large-scalefermentation process of rhKD/APP and found that the best pH is pH3.3±0.2, DObetween 25～30% and the supply speed of methanol is 8.5 ml/h/L initialfermentation volume. The concentration of rhKD/APP in the broth can reach1.0g·L-1.The supernatant of fermentation was diluted with deionized water andadjusted to pH3.8 by sodium hydroxide .The diluted broth was loaded onto columnof SP Sepharose XL cation exchange resin and hydrophobic resin .The absordedrhKD/APP was eluted by 1mol·L-1 NaCl and methanol collected at 280nm.Wefound that this method is simply and reduced cost with the degree of purity beingmore than 95% and the yield coefficient was higher than 70%.3 The protective effect of rhDK/APP on injuried rat hepatocytesThe protective effect of rhKD/APP as a protease inhibitor from humanorigin on hepatocytes and pathologic changes of injuiried liver have not beenreported.So we do some researches of rhKD/APP on protective effects on rathepatocytes and acute hepatic injury and chronic hepatic injury.(1)The effect of rhDK/APP on survival of rat hepatocytesThe purified rhKD/APP inhibits a variety of serine proteases in vitro. Inorder to find the biologic function of rhKD/APP as a protease inhibitor fromhuman origin at the cell level, rhKD/APP was added into rat hepatocytes inprimary culture for 96 hours in the medium supplemented with 2 % new borncalf serum. In contrast ,the medium containing 2 %, 8 % new born calf serumand 10 μg·ml-1 BPTI respectively were used under the same conditions statedabove. It was found that rhKD/APP has the effect on increasing the survival ofrat hepatocytes by observing the morphological feature and cell growth curveanalysis,and still maintained the hepatocyte-like structure. The purifiedrhKD/APP was effective at 2.5 μg·ml-1 and maximally effective at 10μg·ml-1. These results indicated that the rhKD/APP prolongs the survival ofrat hepatocytes.(2)Hepatoprotective effect of rhKD/APP on acute hepatic injuryTo observe the protective effect of rhKD/APP on carbontetrachloride-induced acute hepatic injury and find possible mechanisms ofhepatic injury, Pathologic model of acute hepatic injury in mice caused bycarbon tetrachloride was set up and rhKD/APP with different dosage wereadministrated (ip) to mice at daily on 7 day .Rats were injected with CCL4 at adose of 10 mg?kg-1 body weight as a 0.2% vegetal oil solution and control oneswith the same dose of vegetal oil 1 h after the last injection of rhKD/APP .Allanimals were starved overnight after CCL4 treatment and killed 1 h later afterthe injection the rhKD/APP again.Blood was collected from the femoral arteryunder light ether anaesthesia.Serum was separated by centrifugation for theassay of alanine aminotransferase (ALT) and aspartate transferase (AST)activities.For light microscopy,part of the liver tissue was fixed in 10 %buffered formalin ,embedded in paraffin wax ,and stained with hematoxylinand eosin. The mice treated with rhKD/APP exhibited the amount of ALT,AST in blood serum were decreased. Hepatic tissue damage treated with CCl4showed the extent of the necrosis and hydropic swelling of hepatocytes andinflammatory cell infiltration were more extensive than the group treated withrhKD/APP. The rhKD/APP preventive effect on hepatic injury showed linearrelation to the drug dosage and hepatic injury was alleviated.(3)protective effect of rhKD/APP on chronic hepatic injuryTo observe the protective effects of rhKD/APP on carbontetrachloride-induced chronic hepatic injury and find possible mechanisms ofhepatic injury, pathologic model of chronic hepatic injury in mice caused bycarbon tetrachloride was set up by repeatly injecting of CCL4.Briefly,25%CCL4 mixture with olive oil subcutaneously twice per week at 2 ml?kg-1 bodyweight. Rats injected with olive oil alone served as controls. After 8 weeks ofthe experimental period ,the experiment animals were treated with rhKD/APPat different dose. The control group received equal amounts of saline givenintraperitoneally .After 28 days of the experimental period,the rats were killedand blood was withdrawn from the abdominal aorta for the assay of serumenzyme(ALT,AST,TP,CHE,ALP,ALB,A/G,γ-GT,T-BIL,SA,Hyp)activities.Liver section were taken from each lobe of liver. rhKD/APP can significantlydecresse the activities of ALT AST and the content of Hyp,and ihhibit thedecreasing of ALB ,A/G .The histological changes in the liver showed rattreated with CCL4 revealed destruction of the liver architecture with extensiveaccumulation of connective tissue resulting in formation of continuous fibroticsepta between the central and portal veins .Hepatocyte necrosis ,inflammatorycell infiltration ,voculation, fatty change with cells were seen in the modelgroup.The rat treated with large dose rhKD/APP reduced the liver fibroticformation and hepatocytes necrosis was not seen.The result showed rhKD/APPhas protective effect on experimental liver injury in rats.The rhKD/APP was expressed in 80 L fermentor with optimizingparameters and the broth was harvested with yield of 1.0 g·L-1,Firstly,weconstruct the methods of fermentation of rhKD/APP on large scale andpurifying of rhKD/APP. The rhKD/APP have effect on prolonging the survivalof rat hepatocytes with effective dose at 2.5 μg·ml-1 and maximally effectivedose at 10 μg·ml-1.On the pharmacology research, it was first found thatrhKD/APP has ptotective effect on acute and chronic liver injury in rats.