The Experimental Study On Inducement Of Rat Liver Transplantation Tolerance By Constructing Retroviral Vector Contained FasL Gene

The experimental study on inducement of rat liver transplantation tolerance by constructing retroviral vector contained FasL geneSun Bei-chengWang Xue-haoAbstractObjectives The purposes of this study are (1) to construct the retroviral vector containing rat FasL gene and study FasL-expression in the packaging cell system PA3 17; (2) to transfect the tumor cell with supematant of PA3 1 7/pLXSN-FasL+ in vitro and observe the apoptosis in these tumor cell; (3)to introduce FasL gene into the dendritic cells from the donor, then inject the cells intraperitoneally into the recipient ,observe its survival time of the liver transplantation rat, and search the possible mechanism of immune tolerance induced by transfecting FasL gene therapy.Methods (1)The plasmid pBL-KA15 containing FasL gene was cut with Xho I , and the rat full-length FasL cDNA was obtained at the same time, then subcloned it into the retroviral vector (pLXSN) ligated by T4 DNA ligase,and acquired pLXSN-FasL+ recombinants. Subsequently, pLXSN-FasL+ which in direct inserting were transfected into the amphotropic PA3 17 packaging cells using Lipofectamine, then we acquired G4 18 resistence clones ,and named it PA3 1 7/pLXSN-FasL+ .The polymerase chain reaction (PCR) and RT-PCR were used to detennine whether there were integration of FasL gene and expression of mRNA in the packing cell. The FasL expression on the surface of the packing cell was determined with flow cytometry (FCM). (2)The lymphoma cell strain Jurkat~ Raji~ Daudi, hepatocellular carcinoma cell strain HepG-2 and myeloma cell strain SP2IO were transfected with PA3 I 7/pLXSN-FasL+ in6vbo. The Fas and FasL eXPression on the sdse of these cells wasdMed with fiow Cy'tOmny (FCM), and the aPoPtosis of these cel1swas observed with Annexin V-PI methd, the growth allbihon treatedwith suPeman of PA317/PLXSN-FasL+ tOWed the. Rai. Daudi.HePG2 and SP2/0 ceIls was evaIuated by the cell Proliboon test.(3)Wc liver twlWon in 48 Wister rats Which receiVed SD 1iverwer established by using "the tWoChff tecboque" with somemodifications in cuff tUbe, recipients anaeSthesia and the anastOmoses ofsuPrahepatic vena, pond vein, in~atc vena cava, then, the rats werrandoInly divided into 4 grouPs:@the contrl grou (n=12); @mdrgrou(n==l2) Which treated with Mc cells transfected with mdrl gene;@CsA groUPbe12) haed with cyclOsPorine;@FasL grouP(n=12)treated wdri the deWc cells tafected with FasL gene by tweritoneaIinjection. 4 rats in each gouPs wer killed on the POD3 and POD 7resPectively The liver and periPheral blood were obtaind to detendneliver functiOty W Wssion of Fas. FasL. K-l2 in the gh withsendopantificational RTPCR, the aPoPtosis in hepatocytes and periPherllymPhcyte wer eValUated by W method, the pathological bocterand ultrasboCtUr of the gh were observed through microscoPe andelectron ndcroscoPe.MeanWhile, the post operaon sUrVval time of eachgrou was recorded.ReSulis (lW were FasL direct inserting with single coPy in thecombinants of PLXSMasL+, and it was namd pLXSN/FasL+. The titerof vha in its suPematan reaChe 4.7 X 107CFUffi. It was PrOVed by PCRand RT-PCR tha there wer the integraion of FasL gene and exPression ofInRNA in the paCkin cell. The FasL eXPression with stron intenSity onthe lere of the Packin cell was determined with FCM.(2)The Fasposthe the of the cell sha all. Daudi. Rai. SP2/0 and HePG2detennind wh FCM was 99.8%. 33.9%. 68.6%. 7.2%. 51.7%,7respectively; and the FasL wtssion in the cells ase. Rai. SP2/0 andHePG2 tranSfected wtth FasL gene Was 99.l%. 99.4%. 86.l%. 72.3%,resPectivelyand the aPOPtsis rate was 30.5%' 3l.3%' l2.l%'19.4O/,resPectively;the cell PrOliedn eXPerimen suggested that thegrOwth of tUInr cell sha was reshad Signiflcantiy (3)Afor somemodificationS in the lha forlantation with ..the tWO-cufftedrique",the anhePatc period was reduced sighflcanly the survival rate of liverwtlantation in rats was wtd reMly; the marrw from SD ratwas amPlifled i...