The Related Research Of Immunosuppression In Intermingled Skin Transplantation Induced By B7-H1 Expressed On Auto-keratinocytes

ObjectiveTo explore the possible rloe of the B7-H1 expressed on auto-keratinocytes in the local immunosuppression in the intermingled skin grafting model in vitro(MELC).Methods1.Seperate lymphocytes , epidermic cells and keratinocytes from suckling mice(new born,one to three days old, no hair grown) and then use immunohistochemistry to identify the keratinocytes.2.Establish MELC models of using the lymphocytes , epidermic cells, keratinocytes we separated ,cultured before and in some models auto-keratinocytes are participated to imitate the autologous skin island effects in vitro.3. Isolate keratinocytes after co-cultured in 12h,24h,36h in MELC with auto-keratinocytes or not and examin the B7-H1 expression by flow-cytometry. At meantime, isolate lymphocytes on the same condition, examin PD-1 expression by flow-cytometry..4. Isolate lymphocytes after co-cultured in 12h,24h,36h in MELC with auto-keratinocytes or not,then extract RNA,IL-10,Foxp3,Gata-3 mRNA were detected by Real-time PCR.Results1. Keratinocytes ,epidermic cells and lymphocytes obtained from sulking mice were good in activity,survival,purity. Immunohistochemistry tests of keratinocytes worked well.2. The established MELC by keratinocytes,epidermic cells and lymphocytes displayed well, the co-culturings were stable.3. The B7-H1 expression assumed elevating tendency,it had a predominance diffrence(P<0.05), in MELC with auto-keratinocytes after co-cultured in 12h,24h,36h of isolated keratinocytes by flow-cytometry. And so does the PD-1 expression of isolated lymphocytes in MELC with auto-keratinocytes, it had the same tendency(P<0.05). While in MELC without auto-keratinocytes, both B7-H1 and PD-1 expression did not have any detectable tendency, had no diffrence(P>0.05).4. The IL-10,Foxp3,Gata-3 expression assumed elevating tendency in MELC with auto-keratinocytes after co-cultured in 12h,24h,36h of isolated lymphocytes by using real-time fluorescence quota PCR of extracted RNA, it had a predominance diffrence(P<0.05). While in the MELC without auto-keratinocytes donot have the tendency, had no diffrence(P>0.05).Conclusion1. In the intermingled skin grafting models, B7-H1 expression was induced by auto-keratinocytes and activated T cells,the auto-keratinocytes could enhance the PD-1 expression of T cells by B7-H1.2. Analyze mRNA of T lymphocytes in MELC models,which showed the IL-10 secreting was from lymphocytes but not keratinocytes.3. Analyze mRNA of T lymphocytes in MELC models, Foxp3+Tregs were induced and played an negative role in immune response.4. Analyze the maker gene,GATA-3,of T lymphocytes in MELC models,which turned that Th2 cells held a higher proportion by B7-H1 expression on the auto-keratinocytes.