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OBJECTIVE@#To study placenta-derived mesenchymal stem cells with HLA-G (Human Leukocyte Antigen, HLA-G) positive expression induce Treg (regulatory T cell, Treg) in vitro.@*METHODS@#placenta-derived mesenchymal stem cells were separated from neonatal placenta; PEGFP - N1 -HLA-G plasmid was transfected in placenta-derived mesenchymal stem cells by liposome transfection.The cells were divided into 3 groups including control group, PEGFP-N1 group and PEGFP-N1-HLA-G group, 5 complex walls in each group. Expression of HLA-G protein was detected by Western Blotting; after identification of cells, healthy human peripheral blood CD4 T lymphocytes were cultured with placenta-derived mesenchymal stem cells with HLA-G positive expression, and the ratio of CD4CD25Foxp3Treg in T lymphocytes was accounted.@*RESULTS@#After transfection of PEGFP-N1-HLA-G, the placenta-derived mesenchymal stem cells can express HLA-G protein significantly, compared with the control group and PEGFP - N1 group (<0.01). After HLA-G positive placenta-derived mesenchymal stem cells and CD4 + T lymphocytes were cultured for 24 h, the ratio of CD4CD25Foxp3Treg in T lymphocytes was (16.41±0.94)%. After HLA - G positive placenta-derived mesenchymal stem cells and CD4 T lymphocytes were cultured for 48 h, the ratio of CD4CD25Foxp3Treg in T lymphocytes was (16.46±0.59)% significantly, compared with the control group and PEGFP - N1 group (<0.01).@*CONCLUSIONS@#Placenta-derived mesenchymal stem cells modified by HLA-G gene can effectively induce CD4CD25Foxp3Treg in vitro.
Subject(s)
Female , Humans , Pregnancy , Forkhead Transcription Factors , HLA-G Antigens , Mesenchymal Stem Cells , Placenta , T-Lymphocytes, RegulatoryABSTRACT
Objective HLA-G widely participates in immune tolerance by its combination with immunoglobulin-like tran-scripts IL-2 and IL-4 on the surface of dendritic cells (DCs).The aim of the article was to explore the effects of recombinant adnovirus-mediated HLA-G transfection in macaca mulatta immature dendritic cells on T cell proliferation . Methods Marrow blood was collected from macaca mulattas by the puncture needle after anesthesia .Density gradient centrifugation method was applied in separating mononuclear from the extracted blood on which CD 34+cells were collected and pu-rified by means of immunomagnetic separation .Small doses of cyto-kines were added to get the immature dendritic cells after induced dif-ferentiation of CD34+cells.After the recombinant adnovirus-mediated HLA-G transfection in macaca mulatta immature dendritic cells , observation was done on the viral infection efficiency and western blot was used in detecting the expression of HLA -G in immature den-dritic cells.Taking T cells in macaca mulatta as responders and DCs transfected by recombinant adnovirus -mediated HLA-G as stimu-lators, mixed lymphocyte test was conducted .T cells were divided into 5 groups: mDC group ( mature DCs ) , imDC group ( immature DCs), imDC(L) group(addition of 100 ng/mL lipopolysaccharide after getting imDC at 7th day) , imDC(V) group (imDCs infected by recombinant adnovirus-mediated HLA-G) , imDC( L+V) group ( imDCs infected by recombinant adnovirus-mediated HLA-G along with the addition of 100 ng/mL lipopolysaccharide in culture process ) . Results We obtained the immature dendritic cells and recom-binant adenovirus of HLA-G expressed in these cells .Flow cytometry showed DC purity was up to 92.3 %, imDC purity was up to 72.39%and positive percentage of CD 4+T was greater than 80%.In comparison with imDC group ,the proliferation of stimulated T cells in mDC and imDC(L) groups was obviously intensified (P<0.01).In comparison with imDC(V) group, the proliferation of stim-ulated T cells in imDC, mDC, imDC(L), and imDC(L+V) groups was obviously intensified (P <0.01).In comparison with imDC(L+V) group, the proliferation of stimulated T cells in mDC and imDC(L) groups was obviously intensified(P<0.01). Conclu sion Im-mature DCs infected by recombinant adnovirus can inhibit the proliferation of T cells effectively .
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Objective To investigate the expression level of peripheral blood mononuclear cell(PBMC)human leucocyte anti-gen G(HLA-G)in patients with hepatocellular carcinoma(HCC).Methods The HLA-G mRNA in PBMC from 44 patients with HCC,21 patients with liver cirrhosis and 40 healthy subjects were measured by reverse transcription real time fluores-cent relative quantitative PCR.Results HLA-G mRNA expression level were 1.71±0.39,1.05±0.38 and 1.01±0.47 in HCC group,liver cirrhosis group and healthy control group respectively.HCC group was higher than the other two groups, the difference was statistically significant (F=33.657,P<0.001).The survival rate of HCC patients in HLA-G mRNA high-expression group was lower than HLA-G mRNA low-expression group,the difference was statistically significant (χ2=5.972,P=0.015).Conclusion PBMC HLA-G mRNA in HCC was closely correlated with tumorigenesis.It can proviede a novel diagnosis and research tool for HCC.
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Objective: To study the effect of sHLA-G1 inhibiting xenotransplantation rejection through degrading the releasing of porforin and granzyme by NK92. Methods: The recombinant expression vector pcDNA3-sHLAG1 was transfected into the lymphoblastoid cell line LCL721.221 by the nucleofection methods.The expression of sHLA-G1 in the transfected LCL721.221 was detected by RT-PCR and DOT-ELISA technique.NK cell line(NK92) was used as NK effect cells and the porcine aortic endothelial cells line(PED) as targets.The inhibitive effects of the releasing of porforin and granzyme by NK92 was analyzed by the ?-hexosaminidase release assay.And the cytotoxicity of NK92 was analyzed by MTT methods. Results:sHLA-G1 conferred a significant degrading the releasing of porforin and granzyme by NK92,and protection PED against NK92 medicated lysis,and the rate of NK92 cell cytotoxicity was reduced to 25.5% in contrast to 71.2% in the control group(P