The Role Of Apoptosis In The Heart Transplantation And The Effect Of Gene FasL Transfected By Polyethylenimine On The Allograft Of Rat Heart

Part One The model of rat heterotopic heart transplantationObjective To establish a authentic model of rat heterotopic hearttransplantation. Methods We chose the SD rat as the recipient and the Wistarrat as the donor. The procedure was as follow: The donor heart was procured inadvance. Then the recipient was heparinized. The bole of the right external jugularvein was sheared at the site of more branches and the proximal of it was pull by7/0 polypropylene suture through a self-made canula. The wall of the vein wassheared vertically and was evaginated to encase the wall of the canula, fixed by asilk suture. The right external carotid was prepared in the same way. Results Allthe operations were successful without bleeding, obstruction of the recirculation ordeath of the rats. Conclusions The procedure of the heterotopic hearttransplantation is repeatable, simple and convenient and is applicable to thefollowing investigation.Part Two The role of apoptosis in the rat heart transplantationObjective To explore the role of apoptosis in the hearttransplantation, especially the type of the cell, and the effect of theimmunosuppressant on apoptosis of the cell in the allograft. Methods We chosethe SD rat as the recipient and the Wistar or SD rat as the donor. All the recipientswere divided into 3 groups: group Ⅰ, SD to SD without any treatment; group Ⅱ,Wistar to SD without any treatment; group Ⅲ, Wistar to SD with theadministration of Mycophenolate mofetil (MMF), 10 mg/kg/d from the day prior tothe operation to the 7th day after the operation. The procedures of the operationswere the same as the previous we described. The survival time of the graft, theHE stain of the heart tissue, the expression of Fas, FasL, Bcl-2 and CD4 of theheart tissue, the situ apoptosis of the heart tissue with TUNEL were assessed withstatistic software SPSS 10.0. The difference was significant if the value of P waslower than 0.05. Results 1. All the grafts in groupⅠand Ⅲsurvived longer thanthose in groupⅡ(P<0.05). 2. The expression of Fas and FasL of the heart tissuein groupⅠand Ⅲwere lower than those in groupⅡ(P<0.05), while Bcl-2 higherthan those in groupⅡ(P<0.05). 3. The number of CD4+ cell in groupⅠand Ⅲislower than that in groupⅡ(P<0.05). 4. The apoptotic cell in heart tissue in groupⅠand Ⅲwere fewer than those in groupⅡ(P<0.05). Conclusions Theapoptosis activated by the interaction between Fas and FasL plays an importantrole in the acute rejection in heart transplantation. More of the apoptotic cells arethe cardiocytes and the infiltrating inflammatory cells are just in a small quantity.MMF can inhibit the apoptosis in the graft, which may be a mechanism of itsimmunosuppressive effect.Part Three The effect of gene FasL transfected by polyethylenimine on the allograft of rat heart Objective To explore whether or not that the transfected gene FasLcan induce immunological tolerance in a certain extent in heart transplantationand to determine the efficiency of gene transfection of polyethyleneimine (PEI).Methods We chose the SD rat as the recipient and the Wistar rat as the donor.All the donor hearts were transfected by gene rFasL which were mediated by PEI.The DNA plasmid and the PEI were added to sterile Stanford preservative solutionto get to 150 μl respectively. Then the diluted solution were mingled together forabout 15~30 minutes and the mixture were injected into the coronary arteries ofthe donor hearts to maintain about 20~30 minutes. Finally, the hearttransplantations were performed. All the recipients were divided into 4 groups:group Ⅳ, DNA plasmid (μg): PEI (μl) = 30:60; group Ⅴ, DNA plasmid (μg): PEI (μl)= 20:40; group Ⅵ, DNA plasmid (μg): PEI (μl) = 15:30; group Ⅶ, the donor heartswere transfected only by PEI without gene rFasL. The procedures of theoperations were the same as the previous we described. The survival time of thegraft, the HE stain of the heart tissue, the expression of rFasL of the heart tissue,the situ apoptosis of the heart tissue with TUNEL, the transcription of rFasL mRNAwith RT-PCR and the protein rFasL with Western Blotting were assessed withstatistic software SPSS 10.0. The difference was significant if the value of P waslower than 0.05. Results 1. The survival time of the grafts in group Ⅳwas theshortest (2.9±0.7 days) and that of group Ⅴwas the longest (17.8±0.6 days) (P<0.05). The difference of survival time of the grafts between group Ⅵand Ⅶwasinsignificant (P>0.05). 2. The expression of rFasL in heart got a too high level in ashort time in group Ⅳand got the peak at the 3rd day post-transplantation ingroup Ⅴ, but never exceeded 40% in group Ⅵand Ⅶat the first 3 dayspost-transplantation (P<0.05). 3. The apoptotic index of heart tissue in group Ⅳand Ⅴwere higher than those in group Ⅵand Ⅶ(P<0.05). But more of theapoptotic cells were myocardial cells in group Ⅳand inflammatory cells in groupⅤ. 4. The transcription of rFasL mRNA and the expression of protein rFasL ingroup Ⅴwere stronger than those in group Ⅵand Ⅶ(P<0.05). Conclusions 1.PEI is an efficient vector for gene therapy in the heart transplantation with lowtoxicity. 2. Gene FasL with appropriate concentration can induce immunologicaltolerance in an extent.