Adenovirus-mediated Ex Vivo Gene Transfer Of Human Interleukin-10 Protects Arterial Allograft From Vasculopathy On Rat Model

Orthotopic carotid allograft transplantation was performed by standard microvascular surgical techniques. The ex vivo carotid donor was snaked in Adenovirus-mediated human interleukin-10 solution. Interleukin-10 gene can successfully transfect rat carotid by snaking method. The expression of IL-6,IL-8,TNF-〆was decreased by IL-10 gene,the ischemia-reperfusion injury was lower; IL-10 gene prevented the acute rejection reaction of allograft and relieved impairment of immune reaction, which may be associated with reducing expression of IL-6,IL-8,TNF-〆, attenuation cell apoptosis and relieving impairing of immune reaction . Gene transfer can induce localized expression of Adenovirus-mediated human interleukin-10 by snaking method.Localized and extensive expression of IL-10 gene attenuated inflammatory cell infiltration in the allograft , relieved impairment of immune reaction , which protected allograft vasculopathy on rat model. Gene transfer can induce　localized　expression　of　therapeutic　suppressive　cykokines　within　donor　allograft　that　would　make　this　new　approach　applied　in　the　filed　of　immunosuppressive　therapy　.　Detailed　Abstract　PART　1　　　　　Allograft　vasculopathy　in　an　rodent　model　Objective　:　This　study　is　to　set　up　a　carotid　homeotransplant　model.　Method:　Orthotopic　carotid　transplantation　was　performed　using　wistar　rats　as　donors　and　using　SD　rats　as　recipients　by　microsurgical　technique.　On　1,7,14,30　and　45　days　after　transplantation,　the　allogenic　carotid　was　harvested　and　grafted　carotid　　pathologic　histology　was　examinated.　Result:　After　transplantation,　the　animal　survival　rate　was　more　97﹪.The　patency　is　100﹪　at　time　point　of　14　days　after　transplantation.　Pathologic　histology　showed　that　intimal　hyperplasia　could　be　see　in　all　allograft　carotid　after　4　weeks　transplantation　,　which　demonstrated　that　carotid　transplant　from　Wistar　to　SD　rat.　Conclusion:　 Carotid　　hemeotransplantation　from　Wistar　to　SD　rat　is　a　successful　arterial　allograft　model　which　can　be　used　to　study　allograft　pathologic　mechanism　and　therapeutic　measures.　PART　2　　　　Effect　Of　Adenovirus-mediated　gene　transferof　human　interleukin-10　on　rat　arterial　isograft　ischemia-reperfusion　injury　Objective:　The　goal　of　this　study　was　to　investigate　the　effect　of　Adenovirus-mediated　gene　transfer　of　human　interleukin-10　on　rat　　arterial　isograft　ischemia-reperfusion　injury,and　disccuss　the　mechanism.　Method:　Carotid　of　Wistar　rat　transfected　IL-10　gene　by　snaking　method　was　transplanted　into　carotid　of　SD　rat　with　microsurgical　technique.　SD　rat　donors　and　SD　rat　recipients　were　divided　into　four　groups:　groupA(n=10),receivingsham　operation;　groupB(n=9),receiving　natural　solution;　group　C(n=10),receiving　empty　adenovec; 　 groupD(n=11), 　 receiving 　 adenovec-hIL-10complex.All　allografts　were　harvested　on　8　hours　after　reperfusion　,the　histopathologic　observation　was　carried　out　after　graft　arrest　and　result　were　compared　and　analysed;　Gene　product　expression　in　tissue　was　quantified　by　reverse　transcription-polymerase　chain　reactions　(RT-PCR)and　ELISA;　The　expression　of　hIL-10,　IL-6,　Il-8,TNF-〆　in　　artery　allografts　was　detected　by　using　immunohistochemical　staining　method.　Graft　MDA.SOD.MPO.NO　were　measured.　Apoptosis　cell　death　in　allografts　were　determined　by　in　situ　terminal　deoxynucleotidyl　transferase-mediated　deoxyuridine　triphosphate　nick　end　labeling(TUNEL)and　apoptosis　index(AI)　were　analyzed.　Result:　On　8　hours　after　transplantation　in　group　D,transgene　expression　of　human　interleukin-10　was　detected　by　means　of　both　RT-PCR.　ELISA　and　immunohistochemistry.Expression　scores　of　foreign　gene　significantly　higher　in　Ad.hIL-10　infected　grafts　compared　with　other　three　groups　(P<0.01);The　intimal　imparement　of　groud　D　was　lower　than　other　three　groups;　The　number　of　inflammatory　cell　infiltration　in　artery　allograft　of　group　D　was　less　than　that　of　other　groups,　and　decressed　cell　apoptosis;　IL-6,IL-8,TNF-〆,Fas　and　FasL　were　reduced　in　the　tissue　of　hIL-10　groups(P<0.01);　　MDA,　MPO　were　lower(P<0.05),　but　NO　 , SOD　was　higher　than　the　other　groups(P<0.01);　 Conclusion:　Adenovirus-mediated　human　interleukin-10　gene　transfer　ex　vivo　into　artery　isografts　ameliorates　subsequent　ischemia-reperfusion　injury,　which　was　benefit　for　protecting　allograft　vasculopathy.　PART　3　　　　Effect　of　Adenovirus-mediated　gene　transferof　human　interleukin-10　on　acute　rejection　reaction　of　rat　artery　allograft　Objective:　The　goal　of　this　study　was　to　invertigate　the　effect　of　Adenovirus-mediated　human　interleukin-10　gene　transfer　on　acute　rejection　reaction　of　rat　allograft　arterial　transplantation,　and　discuss　the　mechanisms.Method:　Orthotopic　carotid　artery　transplantation　was　performed　using　Wistar　rats　as　donors　and　using　on　ra　t　arterial　allograft　arteriosclerosis　SD　rats　as　recipients　by　microsurgical　thechnique.　Ex　vivo　donor　was　transfected　Ad.hIL-10　for　30　minutes　by　snaking　method.　On　7　days　after　transplantation,　the　grafts　were　harvested;　Transgene　expression　of　hIL-10　was　detected　by　means　of　both　RT-PCR,　ELISA　and　immunohistochemistry;　The　repression　of　hIL-10,IL-10,IL-2,TNF-〆　in　artery　allografts　were　detected　by　using　immunohistochemial　staining　method;　Graft　IL-2,TNF-〆　were　also　measured　by　ELISA;　Histologic　rejection　score　and　apoptosis　were　also　assessed;　Pathological　morphologic　change　was　analyzed.　Result:　On　7　days　after　transplantation　in　group　Ⅲ,　transgene　expression　of　human　interleukin-10　was　detected　by　means　of　both　ELISA,RT-PCR　and　immunohistochemistry.　The　rejection　score　in　group Ⅲ 　was　significantly　lower　than　that　in　groupⅠ, Ⅱ.　The　expression　of　IL-2,TNF-〆,Fas　and　Fasl　were　reduced　in　groupⅢ　(P<0.01),　and　lower　apoptosis　index　was　also　found　in　groupⅢ(P<0.01).　Conclusion:　The　expression　of　IL-2,TNF-〆　were　successfully　reduced　by　Ad.hIL-10;　Ad.hIL-10　induced　graft　local　immunical　inhabiting　surcuation.　Ad,hIL-10　gene　transfer　ex　vivo　into　rat　artery　allografts　can　inhabit　acute　rejection　reaction　of　allograft　arterial　transplantation.　PART 4 　Effect　of　Adenovirus-mediated　gene　transfer　of　human　interleukin-10　　on　rat　arterial　allograft　arteriosclerosis　Objective:　The　goal　of　this　study　was　to　investigate　the　effect　of　Adenovirus-mediated　human　interleukin-10　gene　transfer　on　rat　arterial　allograft　arteriosclerosis,　and　discuss　the　mechanisms.　 Method:　Orthotopic　carotid　artery　transplantation　transfected　Ad.hIL-10　was　performed　using　Wistar　rats　as　donors　and　using　SD　rats　as　recipients.　On　45　days　after　transplantation　in　group　3,　the　allografts　were　harvested.　The　allografts　pathologic　changes　were　observed　and　the　average　intimal/medical　ratio　of　the　allografts　were　measured;　Transgenen　expression　of　human　interleukin-10　was　detected　by　means　of　both　RT-PCR,ELISA　and　immunohistochemistry;　The　expression　of　hIL-10,IL-1,IFN-г,ICAM-1,VCAM-1,TIMP-1,〆-actin,　Fas　and　Fasl　were　detected　by　using　immunohistochemical　staining　method;　and　apoptosis　were　also　assessed.　Result:　On　45　days　after　transplantation　in　group　3,　RT-PCR,　ELISA　and　immunohistochemistry　demonstrated　the　allograft　hIL-10　gene　transcripts　in　the　allografts.　Immunohistochemical　examination　of　Ad.hIl-10　infected　grafts　(grou　3)　confirmed　successful　hIL-10　gene　transfer　expression　in　vascular　endothelial　cells　and　smooth　muscle　cells;　Expression　scores　of　foreign　gene　were　significantly　higher　in　Ad.hIL—10　infected　grafts　compared　with　other　two　groups(P<0.01);　The　intimal　hyperplasia　and　the　midle　hyperplasia　　ratio　were　significantly　lower(P<0.01);　Grafts　apoptosis　index(AI)　in　groupⅲ　were　less　than　the　other　groups　by　TUNEL(P<0.01);　The　　expression　of　IL-1,IFN-г,ICAM-1,VCAM-1,　〆-actin,　Fas　and　FasL　were　reduced　in　group　3　(P<0.01),　but　TIMP-1　was　higher　than　the　other　groups　(P<0.01);　Conclusion:　These　data　demonstrate　that　gene　transfer　by　snaking　method　of　recombing　adenovirus　vector　encoding　immunosuppressive　cytokines　hIl-10　to　carotid　allograft　was　efficient　and　feasible,　and　protected　carotid　allograft　arteriosclerosis,　which　would　make　this　approach　widely　clinically　applicable.