本文選題：人類白細(xì)胞抗原-G基因　切入點(diǎn)：T細(xì)胞　出處：《醫(yī)學(xué)研究生學(xué)報(bào)》2017年01期

【摘要】：目的人類白細(xì)胞抗原-G(HLA-G)通過(guò)與樹突狀細(xì)胞(DC)表面的免疫球蛋白樣轉(zhuǎn)錄體(ILT2)和ILT4(CD85d)結(jié)合,廣泛參與機(jī)體的免疫耐受的過(guò)程。探討經(jīng)HLA-G重組腺病毒載體感染后的恒河猴未成熟樹突狀細(xì)胞刺激T細(xì)胞的增殖作用。方法麻醉恒河猴獲取新鮮骨髓血,采用密度梯度離心法,分離獲得的單個(gè)核細(xì)胞行免疫磁珠法獲得CD34+細(xì)胞。添加小劑量細(xì)胞因子聯(lián)合誘導(dǎo)分化CD34+細(xì)胞,從而獲得樹突狀細(xì)胞。介導(dǎo)HLA-G的重組腺病毒經(jīng)過(guò)轉(zhuǎn)染未成熟樹突狀細(xì)胞后,檢測(cè)病毒感染效率,Western blot檢測(cè)HLA-G在未成熟樹突狀細(xì)胞中的表達(dá)。以恒河猴T細(xì)胞作為反應(yīng)細(xì)胞,將介導(dǎo)HLA-G的重組腺病毒轉(zhuǎn)染的不同狀態(tài)下的DC為刺激細(xì)胞,進(jìn)行混合淋巴細(xì)胞實(shí)驗(yàn)。設(shè)置5組:即成熟樹突狀細(xì)胞組(mDC組);未成熟樹突狀細(xì)胞組(imDC組);imDC(L)組:在第7天成功獲得imDC后加入脂多糖100 ng/m L;imDC(V)組:重組腺病毒介導(dǎo)HLA-G感染的imDC,方法同前;imDC(L+V)組:重組腺病毒同樣轉(zhuǎn)染imDC,同時(shí)在培養(yǎng)過(guò)程中添加100ng/m L的脂多糖。根據(jù)DC與T細(xì)胞不同比例(1∶5、1∶10、1∶20、1∶40)計(jì)算各組刺激指數(shù)(SI)。結(jié)果成功培養(yǎng)獲得恒河猴未成熟樹突狀細(xì)胞,HLA-G在該細(xì)胞中表達(dá)。流式細(xì)胞術(shù)檢測(cè)結(jié)果發(fā)現(xiàn)DC的純度高達(dá)92.3%,imDC組則高達(dá)72.39%,CD4+T細(xì)胞陽(yáng)性率純度80%�；旌狭馨图�(xì)胞實(shí)驗(yàn)分析DC刺激T細(xì)胞增殖結(jié)果表明,不同DC與T細(xì)胞比例(1∶5、1∶10、1∶20、1∶40)下,imDC組SI為1.63±0.03、1.52±0.04、1.39±0.03和1.21±0.01;mDC組SI為2.23±0.05、2.01±0.03、1.65±0.02和1.54±0.05;imDC(L)組SI為2.25±0.04、1.96±0.02、1.62±0.03和1.48±0.01;imDC(V)組SI為1.46±0.04、1.33±0.02、1.20±0.04和1.04±0.02;imDC(L+V)組SI為1.67±0.03、1.59±0.04、1.38±0.03和1.24±0.03。與imDC組比較,mDC組、imDC(L)組SI明顯增高(P0.01);與imDC(V)組比較,imDC組、mDC組、imDC(L)組、imDC(L+V)組SI明顯增高(P0.01);與imDC(L+V)組比較,mDC組、imDC(L)組SI明顯增高(P0.01)。結(jié)論重組腺病毒介導(dǎo)HLA-G轉(zhuǎn)染的恒河猴未成熟樹突狀細(xì)胞能夠有效抑制恒河猴T細(xì)胞的增殖。
[Abstract]:Objective Human leukocyte antigen (HLA-GN) binds to immunoglobulin-like transcripts (ILT2) and ILT4 (CD85d) on the surface of dendritic cells (DC) to participate in the process of immune tolerance.To investigate the proliferation of T cells stimulated by immature dendritic cells of rhesus monkey infected with HLA-G recombinant adenovirus vector.Methods fresh bone marrow blood was obtained from anesthetized rhesus monkey and CD34 cells were obtained by density gradient centrifugation.Dendritic cells were obtained by adding low dose cytokines to induce differentiation of CD34 cells.After the recombinant adenovirus mediated HLA-G was transfected into immature dendritic cells, the viral infection efficiency was detected. Western blot was used to detect the expression of HLA-G in immature dendritic cells.The T cells of rhesus monkey were used as reaction cells and DC transfected with recombinant adenovirus of HLA-G was used as stimulator cells to carry out mixed lymphocyte experiment.璁劇疆5緇，

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