A standard microlymphocytotoxicity assay was used to test the sera for HLAantibodies in the patients receiving multitransfusions and to identify their speci-ficity,and at the same time the platelet-associated antibodies were detected.As a result,the dectable rate of HLA antibodies was 39.14% (119/304),of which24.37% (29/119) had specificity;the positive rat platelet-associated antibodieswas 39.83% (47/118).The frequency of positive incidence of HLA antibodiesincreased with the number of transfusions and donors,having a significant differ-ence.The nonhemolytic reaction to transfusion was associated with HLA antibodi-es.The frequency of positive incidence of HLA antibodies was the highest whenthe patients were transfused with concentrated granulocytes.There were takensome measures to prevent and reduce the transfusion reaction.

Objective: To optimize and evaluate the modified RNA-tailing and primer-extension RT-PCR method in relative quantification of microRNAs (miRNAs) in several kinds of tissues. Methods: Small-sized RNAs (

Objective: To investigate the non-specific and inaccurate amplification in cases of highly similar sequences among family members and the length heterogeneity of mature microRNA ( miRNA) ,and find a condition that discriminates maximally among similar miRNA family members and detects the accurate expression level of miRNAs. Methods: Primers with their mismatches and/or 3' end at different positions were designed. Amplification efficiencies were compared using matched and various mismatched primers by RNA-tailing and primer-extension RT-PCR at different annealing temperatures. Expression levels of several miRNAs in mouse brain were compared using miRNA specific primers with different termini. Results: Raising annealing temperatures 12℃-14℃above the T_m of the primers maximally increased amplification specificity without sacrificing sensitivity. Primers designed with their termini on or near variant positions could efficiently discriminate between miRNA isoforms. Using primers that terminated before the end of the mature miRNA did not miss the detection of shorter mature miRNA and provided accurate expression levels. Conclusion: Careful primer design and higher annealing temperature can increase specificity and accuracy of real time PCR miRNA detection.

Objective To detect and determine the expression and significance of MHC class Ⅰ chain-related gene A/B (MICA/B) and membrane MIC molecules (mMIC) on the bone marrow mononuclear cells (MNC) of patients with acute leukemia (AL). Methods Expression of MICA/B gene was detected by semi-quantitative reverse transcriptaso polymerase chain reaction (RT-PCR) in MIC-pesitive K562 cell line, bone marrow MNC from 10 healthy people and 69 cases of acute leukemia (AL). Expression of mMIC was detected by Western blotting. The differences of the expression of MIC gene and mMIC between AML and ALL were compared. The prognosis was determined by chromosome type between patients with mMIC+ and mMIC-. Results The expression of MIC gene and mMIC could not be detected in healthy people. The expression rate of MICA gene was 49.28% and the MICB gene was 42.03% and the mMIC was 34.78% in patients with AL. In AML group, the expression rate of MICA gene was 60.00%, and the expression rate of MICB gene was 53.33%, and the expression rate of mMIC was 44.44%. But in ALL group, the expression rate of MICA gene was 29.17%, of MICB gene 20.83%, and of mMIC 16.67%. The expression of MICA/B gene and mMIC in AML group were higher than that in ALL group (P<0.05). The prognosis of patients with mMIC+ is better than the ones with mMIC-. Conclusion The up-regnlation of MIC gene and mMIC in bone marrow MNC from patients of AL may have some relationship with the occurrence of AL The expression of MIC gene and mMIC is high in AML and low or devoid in ALL, which would be an possible mechanism that ALL cells were easy to escape killing from NK and CTL cells. Determined by chromosome type, the prognosis of AL with mMIC positive was better than the ones with mMIC negative. MIC might be one of the factors to determine the prognosis of AL.

Objective: To study the immunotherapeutic effect on the esopgageal adenocarcinoma mediated by gp96-peptide complexes isolated from the same kind of tumor. Methods: gp96- peptide complexes were purified from nude mice tumors burdened by subcutaneous injection of human esophageal adenocarcinoma cell line SEG-1 . gp96-peptide complexes were carried by the dendritic cells(DC) induced from human peripheral blood mononuclear cells to prepare gp96-DC vaccine. The proliferation of lymphocytes was tested with trypan-blue stain. The quantity of interferon-?(IFN-?) released from cytotoxic T lymphocytes (CTL) was detected with ELISA method. The killing effect of CTL on target cell SEG-1 was measured with MTT. Results: We obtained 120 ?g gp96 from 55 g tumor tissue. DC, gp96, and gp96-DC all could elicit the proliferation of lymphocytes and make them becoming into CTL which released IFN-? and showed different degrees of killing effect on target cell SEG-1. gp96-DC has the strongest eliciting effect among them. At the ratio of E(effect) to T(target) as 40∶1,the killing rate was 68%.No significant difference between the effects of CTL induced by DC alone and of lymphocytes without specific antigen on SEG-1 and K562 cells. Conclusion: The gp96-peptide complexes from tumors can improve the effect of eliciting lymphocyte proliferation of DC and make the lymphocyte becoming into CTL more effectively.These CTLs show prominent killing effect on the target tumor cells.

Objective:To examine global expression levels of microRNAs(miRNAs) in mouse cerebrum and to provide an important basis for detailed studies of individual miRNAs,their target genes,the miRNA-related regulatory networks in the mammalian central nervous system,and their implications in diseases.Methods:Low molecular weight RNA from cerebrum of five C57BL/6J mice were tailed and reverse transcribed by extended RT-primer.miRNA primers were carefully designed and arrayed on plates according to the Tm of each primer.PCR was carried out at different annealing temperatures using a gradient real-time PCR instrument.The relative expression level of each miRNA was calculated using 5sRNA for normalization.Results:Among the 285 miRNAs detected,260 were positive with varying abundance.Their frequency distribution was approximately a normal distribution.The expression levels of most miRNAs were in accordance with previously published results by microarray.However,the positive rate was higher than that detected by microarray.miRNAs originating from the same hairpin precursors expressed at similar or significantly different levels.Clusters of proximal miRNAs were similar or quite different in abundance.It is suggested that the fate of miRNA after transcription determined their abundance.Conclusion:Using the RNA-tailing and primer-extension PCR array method,we obtained expression profile of miRNA in mouse cerebrum,especially the relative expression data of many low abundant miRNA in mouse cerebrum,which will be of special help for studying the fine-tuning function of low-level miRNAs.

Objective To study the effect of homeobox genes CDX1 and CDX2 on the phenotype of esophageal adenocarcinoma cells.Methods Esophageal adenocarcinoma cell line SEG-1 was transfected with CDX1 or CDX2 cDNA.The morphology,growth rate,division index and tumorigenicity were analyzed.Results The expression of CDX1 or CDX2 leaded to occurring adeniform-like manifestation.The growth rate,division index and tumorigenicity were reduced,especially by CDX2.Conclusion CDX1 and CDX2 all could increase differentiation of esophageal adenocarcinoma cells and reduction of its malignancy.

Objective To observe the preventive effects of Tripterygium hypoglaucum(Level)Hutch(THH)extract on T lymphocyte subpopulations in mice with graft-versus-host disease(GVHD)and to explore its protective mechanism.Methods 2?107 bone marrow cells mixed with 2?107 spleen cells from C57BL/6 mouse were transplanted into the myeloablative irradiated inbred BALB/c mouse.The experiment groups were designed as follows:model control group,cyclosporine A(CsA,10 mg?kg-1?d-1)group,THH(400 mg?kg-1?d-1)group,combination group(CsA 5 mg?kg-1?d-1 + THH 100 mg?kg-1?d-1).The ocurrence of GVHD was assessed by signs of weight loss,diarrhea,ruffled fur,hunched posture and histologic changes of skin,liver and small intestines.Chimerism was detected by monitoring the H-2b molecule in bone marrow cells of recipient mice with flow cytometer.The percentages of CD+3CD+4 and CD+3CD+8 T cells in peripheral blood were detected by flow cytometer.Results The survival time of CsA group,THH group and combination group were prolonged as compared with that of the model control group(P

Objective To investigate the relation and difference of expression phase between classⅡtransactivator (CⅡTA) and HLA-DR antigens after IFN-? incubation, so as to investigate the potential effect of the class Ⅱ trasactivator (CⅡTA) in graft-versus-host disease(GVHD). Methods T lymphocyte from peripheral blood of health was incubated with IFN-? for 1 000 U/ml. RT-PCR was used to detect CⅡTA mRNA and Western blot was used to explore HLA-DR antigen in various time periods. Then Stat1? antisense oligonucleotides (AS) were given to inhibit the expression of CⅡTA, CⅡTA mRNA and HLA-DR antigen were tested again at peak point. Results CⅡTA mRNA was detectable 5 h after IFN-? treatment and peaks at 14 h; HLA-DR protein was detectable 28 h after IFN-? treatment and peaks at 52 h. The expression of CⅡTA mRNA and HLA-DR protein was lower in AS groups than that in control groups. Conclusion CⅡTA expression was positively correlated to HLA-DR expression, and earlier than the HLA-DR expression after IFN-? incubation. CⅡTA might be used as an early predicting marker of GVHD.

Objective:To investigate the expression of CD28 costimulatory molecule on human lymphocytes inhibited by siRNA.Methods:Three different siRNA (siRNA 1,siRNA 2,siRNA 3) were designed and synthysized and transfected into freshly isolated human lymphocytes with cationic liposome.At 24,48 and 72 h post transfection,the changes of CD28 expression were detected by flow cytometry,and the changes of CD28 mRNA levels were determined by semi quantitative RT PCR.Results:Different siRNA showed different reduction in CD28 expression.At 48 h post transfection,the degrees of reduction with siRNA 1,siRNA 2 and siRNA 3 were 22 10%?1 63%,73 50%?1 02% and 42 90%?0 89% respectively compared with the control ( P