| BackgroundIt is a effective measure for many diseases by bone marrow transplantation(BMT) or hematopoietic stem cells transplantation(HSCT). badly short of HLA matched donors is the hindrance,it is often failed in HLA mismatched BMT because graft versus host disease(GVHD).In recent years researches on killer cell inhibitory receptors(KIR) have indicated a new way to reduce or aviod GVHD in BMT.Killer cells including natural killer cells (NK) and cytotoxic T lymphocytes(CTL) are impotant effective cells in GVHD .KIRs expressed on NK and T cells are key moleculars in immunity cytotoxicity regulation .When binding with corresponding HLA I moleculars ,negative signal is transduced and the cytotoxity of NK and T cells are abrogated/down regulated. CD158 ,including CD158a and CD158b,is a member of KIR superfamily.The specfic ligands of CD158 are human leukocyte antigen C(HLA-C) expressed on target cells.The Caucasian CD158 gene were cloned by Long EO Laboratory in USA.Some reseaches has been made on the singal system.but no reports about CD158 gene in China.In our experiments CD158 gene of Chinese were cloned from normal person, Constructed and Identificated of Recombinant Defective Retroviral Vector,transfected into lymphocytes of donors and got expressing,observing killed effect by mixed lymphocyte reaction(MLC).The molecular mechanism of the interaction between CD158/HLA-C was discussed,Which can provided experimental data for prevention and cure GVHD. ObjectiveTo clone and identify CD158 gene which encode natural killer cell inhibitory receptor.expressing on lymphocytes and to study the function of them by gene transfection and mixed lymphocyte reaction(MLR)Methods1. Having isolated peripheral blood mononuclear cells(PBMCs) from peripheral blood of normal person by Ficoll-Hypaque density gradient centrifugation,Total RNA were extracted according to Trizol kit(GIBCO/BRL). Identified by improved gel electrophoresis.The CD158 gene were amplified by RT-PCR and recombinant cloning vector pSPORT1-CD158 was constructed and sequenced after the enzyme digestion.2. Construction a recombinant defective retroviral expression vector carrying CD158b gene from pSPORTl-CD158b,identified by the enzyme digestion and PCR.3. The recombinant retroviral expression vector was packaged by PT67 cells,screening stable virus-producing cell lines,infecting donors lymphocytes (erythrocyte and platelet,besides monocytes are all wiped off).The expression rate of CD158b receptor was detecting by Flowcytometer(PE-CD158a Mab and PE-CD158b) after transfection.4. MTT method was applied to assay the killing effects of donors lymphocytes.5. All data were dealt with SPSS 10.0(One-Way ANOVO Test) Results1. The result of RNA electrophoresis indicated that the total RNA was not broken down , A260/A280=1.92 also indicated the total RNA was pure.It can be amplified by RT-PCR.2. The gene segment amplified by RT-PCR was the same as reported in Gen Bank(siniilarity of base pair>95%).3. The expression rate of CD158b receptor (CD158b gene was studied)was (45.72 *0.58)% for lymphocytes transfected with pMSCVneo-CD158b; (18.05 * 0.75)% for lymphocytes transfected with pMSCVneo and (19.32 * 0.31)% for untransfected control.(F=655.97,P=0.0003).4. MTT assay indicated OD570 in group of pMSCVneo-CD158b transfceted donor lymphocytes for 24 hours(0.654 ±0.052) , (0.726 ±0.078) for transfceted donor lymphocytes 10 days late, pMSCVneo transfceted donorlymphocytes for 24 hours (0.387±0.038),No vector transfecting(0.417±0.039),( F=78,266,P=0.009),Conclusions1. DNA sequencing showed we have gotten the human CD 158 complete cDNA.Improved method on RNA electrophoresis is simple and effective.One method for cloning the CD 158 gene was found.2. The expression rate of CD158b showed that The retro viral expression vector pMSCVneo was effective for transfection,Which could be used for gene transfer. It also showed CD158b could express effectively on lymphocytes.3. In the...
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