To Study The Relationship Between The Gene Expression Of HLA-A In Peripheral Blood Leukocytes And The Immune State Of Recipients

Object To investigate the feasibility of HLA-A, -B in peripheral blood lymphocyte ( PBL ) from kidney transplantation patients as host immune index,especially reject reaction index by establishing the method of detecting HLA-A non-polymorphism locus using Reverse Transcriptase- polymerase chain reaction (RT-PCR) and compare it with the ratio of CD4/CD8 and concentration of immunosuppressant(FK506).Methods According to HLA-I gene sequence announced in GenBank, the special primers targeting for the conservation districts in ex on 4, 5 of HLA-A were synthesized and screened, then amplificated by one-step RT-PCR. 30 kidney transplantation patients within 3 months after surgery (follow-up of 10 patients), and 61 normal persons as control were included in the study. Total RNA of PBLs from patients and healthy volunteers were extracted, and the amplified fragments were analyzed by Bandscan software after agarose gel electrophoresis to semi-quantify the expression of HLA-A. Referring to relevant documents the specific primers of HLA-B were synthesized to examine the expression of HLA-B mRNA by RT-PCR method in 5 follow-up transplant patients and 20 normal control, and compared with the expression of HLA-A. The ratio of CD4/CD8 and the concentration of FK506 were also measured by flow cytometry(FCM) and enzyme- linked immunosorbent assay (ELISA).Results1. The positive expression rate of HLA-A mRNA in renal transplant patients is 29%(20/70), significantly lower than the control (82%, P<0.001). 2 of 10 follow-up patients occurred acute rejection, and during the rejection period the expression ofHLA-A increased. And distinct patient has different result at the same period after transplantation, with respect to rejection reaction and infection.2. The positive expression rate of HLA-B mRNA in renal transplant patients with follow-up study is significantly lower than normal control (P<0.05). The result is consistent with that of HLA-A(P>0.05).3. The subset of T lymphocyte in peripherial blood has statistical difference between transplantation patients and normal control(P<0.01).The percent of distinct subset and the ratio of CD4/CD8 in transplant patients is significantly lower than normal control. And the expression of HLA-A correlates with the ratio of CD4/CD8 in transplantation patients(P<0.05).4. The concentration of FK506 has correlation with the expression of HLA-A in transplantation patients (PO.05).Conclution1. The method of detecting HLA-I gene by RT-PCR was succefully established.2. Compared with normal control, the expression of HLA-A ^ -B in peripheral blood lymphocyte from transplant patients significantly decreased. At the occurrence of reject reaction the expression of HLA-A> -B in PBL increased, suggesting the probability of HLA-I in PBL as the diagnosable index of reject reaction in the early stage of transplantation. And the expression intensity has correlation with the concentration of immunosuppressant.3. HLA-I has double role, extron 2^ 3 represent the polymorphism of HLA, determing the occurrence of rejection reaction; extron 4^ 5 reflect host immune state, implying the intensity of rejection reaction.