Adenovirus-mediated OX40Ig Gene Transfer Induces Long-term Survival Of Liver Allografts In Rats

Objectives(1) To set up a successful model of orthotopic liver transplantation in rats by modified two-cuffed technique..(2) To set up an acute rejection model of orthotopic liver transplantation in rats by modified two-cuffed technique.(3) To construct a recombinant adenovirus vector pAdeasy-1-pAdTrack-CMV-OX40Ig (AdOX40Ig).(4) To determine the influence and infection efficiencies of different titers of AdOX40Ig recombinant adenovirus on the growth of human hepatocyte HL-7702,and select out the best titer to study on the biological function of the virus.(5) To discuss the role and regime of adenovirus-mediated OX40Ig gene in inducing long-term survival of liver allografts in ratsMethods(1) Orthotopic liver transplantation was conducted in 130 pairs of SD rats by modified two-cuffed technique.The first 40 pairs are the pre-experimental group,the middle 60 pairs are the experimentally skilled group,and the last 30 pairs are called the experimentally consummate group.The successful operative rate,the postoperative survival rate and the operative complications were observed.(2) Thirty cases of Orthotopic liver transplantation was performed in Lewis to BN rats through the modified two-cuffed technique,and all rats were randomly divided into 2 equal groups.One was experimental group which were not specially treated,and the other was contrast group which took gastric perfusion with 0.2mg/(kg.d) FK506 during 1～7 days after operation.Each time on the 4th,5th and 10th days after transplantation,3 rats in either group were killed,whose blood and liver samples were collected while the other receptor rats' blood samples were drawn from their lingual vein.Blood samples were used for examining cytokine and biochemical indicators.Liver lobes were applied to detect pathological changes and liver cell apoptosis.The 6 survival rats in each group were used for recording postoperative living status and survival time.(3) After double digestion,hOX40,hIgG1 Fc fragment and pAdTrack-CMV vector were connected for gene recombination,then transferred into competent E.coli cells and generated pAdTrack-CMV-hOX40Ig transfer plasmid.It was verified by PCR amplification,double-enzyme digestion and DNA sequencing,the correct plasmid named pAdTrack-CMV-OX40Ig(pTOX40Ig).After linearization of transfer plasmid PTOX40Ig,the plasmid was co-infected with pAdeasy-1 adenovirus vector into competent BJ5183 cells by the method of calcium chloride.Then the cells were coated on the tablet containing kanamycin.Correct clone was selected by extraction of plasmid,and thereafter PCR and restriction enzyme digestion by Pacâ… for verification.The correct recombinant plasmid pAdOX40Ig was transfected into 293A cells by using Lipofectamine 2000 after digested by Pacâ… .On the third day after tansfection,the fluorescence was observed under fluorescence microscope.The recombinant adenovirus AdOX40Ig was also identified by Western blot.(4) Recombinant adenovirus AdvOX40Ig infected HL-7702 cells at different titer such as 1,10,25,50,100,200 MOI.Cell morphology of infected HL-7702 was observed under light microscope,and the green fluorescent was observed under the fluorescent vision. AdvOX40Ig recombinant adenovirus and mock adenovirus were infected into HL-7702 cells at 50MOI dose respectively.Adenovirus-mediated exogenous gene OX40Ig expression in HL-7702 cells was detected through indirect immunofluorescence by immunofluorescent staining kit.ELISA method was also used to determine the concentration in the culture supernatant after HL-7702 liver cells infected with adenovirus AdvOX40Ig.(5) Forty cases of Orthotopic liver transplantation was performed in Lewis to BN rats through the modified two-cuffed technique,and all rats were randomly divided into 4 equal groups:contrast group(neither donors nor receptors were treated),AdEGFP treated group (donor liver carried 2ml adenovirus AdEGFP[1×10⁹pfu/ml]after it was excised during operation,AdOX40Ig treated group(donor liver carried 2ml adenovirus AdOX40Ig [1×10⁹pfu/ml]after it was excised during operation),and FKS06 treated group(gastric perfusion with FK506(0.2mg·kg^-1·d^-1) after operation).On the 7th day after operation,3 BN rats in each group were killed.Blood was then drawn from the inferior caval vein to detect the liver funtion.Liver was excised to detect pathological changes and liver cell apoptosis.Spleen cells were collected to undergo mixed lymphocyte reaction(MLR) with the Lewis rats and the third strain F344 rats' spleen cells. Three repeated holes were prepared in the MLR process between BN rats and Lewis rats among which each was mixed with 20μl(1 IU/μl) Recombinant IL-2.With the remaining blood of the 4-group killed BN rats and the blood preoperatively drawn from lingual vein, the content of IFN-γ,IL-2,IL-4 and IL-10 was detected by double antibody sandwiched ABC-ELISA procedure.The survival rats were used to detect live range,and their blood was collected from caudal vein on the day virus was planted and on the 1st,3rd,7th,10th, 14th,21th and 28th days after that treatment.The OX40Ig protein expression level was assayed ELISA procedure. Results(1) The successful operative rate and the 7-day survival rate were respectively 27.5% and 0 in the pre-experimental group,75.0%and 40.0%in the experimentally skilled group, and 93.3%and 86.7%in the experimentally consummate group.(2) The successful operative rate was 93.7%(30/32).The experimental group of rats exhibited obvious acute rejection on the 7th～10th days after operation.The live range was 9.17±1.17 days for the experimental group and 50.17±8.50 days for the contrast group.The liver function of the experimental goup was obviously better than that of the control group. As for the experimental group,the content of IFN-γrand IL-2 on the 7th postoperative day was significantly more than that in the preoperative time,vice versa for IL-4 and IL-10.As for the contrast group,the result was opposite.The postoperative hepatic pathological results showed that medium and severe cellular rejection turned up respectively on the 7th and 10th postoperative days in the experimental group while in the contrast group mild cellular rejection turned up on the same days.The liver cell apoptosis was distinctively more severe in the experimental group than that in the contrast group.(3) By enzyme digestion and DNA sequencing,the sequence of recombinant adenovirus vector pAdOX40Ig was proved to be correct.In infection of 293A cells,could be seen on the third after transfection.After several rounds of infection of recombinant virus,testing titer reached 10¹⁰pfu/ml.A strip of 48.7kD could be seen from Western blot on identification of recombinant AdOX40Ig.(4) Under light microscope,recombinant adenovirus infected HL-7702 cells at 1,10,25,50,100 MOI of different doses were morphologically normal,grow well,and infected cells at 200MOI were round shrinkage and off shown clear signs of cell toxicity. At fluorescence Perspective,the green fluorescence were almost all could be observed in recombinant adenovirus infected HL-7702 cells at 10,25,50,100,200 MOI. AdvOX40Ig infected HL-7702 cells could be detected with the Cy3 red fluorescence, while the mock virus infection group and non-infection group HL-7702 cells couldn't be seen in red fluorescence.Determination of ELISA results showed that,AdOX40Ig infection group was significantly higher than non-infected group and AdEGFP infected group.(5) The living range of rats in the AdOX40Ig and FK506 treated groups was obviously longer than that in the control and AdEGFP treated groups,while the hepatic function of the previous groups was worse than that of the latter groups,the previous' hepatic cellular rejection was obviously milder than the latter's,and the previous' liver cell apoptosis was less than the latter's.The content of IFN-γand IL-2 in the blood after operation was obviously lower than that before operation(P＜0.05),vice versa for the content of IL-4 and IL-10(P＜0.05) in the AdOX40Ig and FK506 treated groups.As for the contrast and AdEGFP treated grous,the the postoperative content of IFN-γand IL-2 was obviously higher than before operation(P＜0.05) while the postoperative content of IL-4 and IL-10 was obviously lower than before operation P＜0.05).The CPM indexes of the AdOX40Ig and FK506 treated groups were obviously higher than that of the contrast and AdEGFP treated groups.With respect to the MLR reaction between BN rats and Lewis rats,the CPM indexes of AdOX40Ig and FK506 treated groups were significantly higher when the MLR was mixed with recombinant IL-2 than when the MLR was not mixed with recombinant IL-2.As for the MLR reaction between BN rats and F344 rats,there's statistically no meaning in comparison between each other about the CPM indexes of contrast,AdEGFP and AdOX40Ig treated groups.Nevertheless,the CPM indexes of the three groups were significantly higher than the FK506 group.Conclusions (1) The skilled and consummate surgical operative technique is key to successful operations.Shortening the anhepatic period efficiently ensures the long-term survival after operation.(2) The acute rejection reaction between Lewis rats as donors and BN rats as receptors was fierce and stable,and thereof,an ideal acute rejection model for liver transplantation in rats.(3) The recombinant adenovirus pAdOX40Ig was successfully constructed and the titer of virus was 10¹⁰pfu/ml.(4) Our results showed that different titers of virus such as 10,25,50,100MOI were not only non-toxic to cells but with high infection efficiency ahnost up to 90%.All these showed that 10MOI maybe the best infection titer.The results of indirect immunofluorescence test and ELISA showed that adenovirus induced exogenous OX40 gene expressed successfully in HL-7702 cells.(5) The donor liver with OX40Ig treatment had the function of countering acute rejection,just like FK506.Specifically,the rats with AdOX40Ig treatment had a longer living range,milder hepatic cellular rejection,less hepatic cellular apoptosis,lighter harm to hepatic function,immunoreaction toward Th2 shift,and reversibility of depressed mixed lymphocyte reaction by IL-2.Unlike FK506,the donor liver with AdOX40Ig treatment could depress mixed lymphocyte reaction induced by activating cells of the same strain rats but could not depress mixed lymphocyte reaction induced by activating cells of rats of different stains.