【摘要】：目的探討T細(xì)胞活化過(guò)程中表面Tn抗原的表達(dá)狀況與Cosmc轉(zhuǎn)錄及蛋白表達(dá)水平之間的關(guān)系。方法30例健康志愿者,男15例,女15例,平均年齡(34±8.9)。抽取志愿者外周靜脈血,肝素抗凝,采用Ficoll密度梯度離心法分離單個(gè)核細(xì)胞(peripheral blood mononuclear cell,PBMC),免疫磁珠法分選純化的CD3+T細(xì)胞,分別采用T細(xì)胞活化劑Cocktail、CD3/CD28Dynabeads刺激12、24、36、48、60、72h。流式細(xì)胞術(shù)檢測(cè)兩種活化劑刺激活化前后不同時(shí)間點(diǎn)CD3+T細(xì)胞中Tn+細(xì)胞的百分率及Tn抗原平均熒光強(qiáng)度,采用RT-PCR和Western blot檢測(cè)兩種活化劑刺激活化前后不同時(shí)間點(diǎn)T細(xì)胞中Cosmc轉(zhuǎn)錄及蛋白表達(dá)狀況,采用熒光分析法檢測(cè)T-synthase活性,應(yīng)用SPASS13.0軟件進(jìn)行統(tǒng)計(jì)學(xué)分析,闡明活化T細(xì)胞表面Tn抗原動(dòng)態(tài)變化與Cosmc之間的關(guān)系。結(jié)果 Cocktail、CD3/CD28Dynabeads均能促使活化T細(xì)胞表面表達(dá)Tn抗原,以及Tn抗原表達(dá)的平均熒光強(qiáng)度隨活化時(shí)間的延長(zhǎng)先升高后下降。CD3/CD28Dynabeads活化組Tn+細(xì)胞百分率的峰值出現(xiàn)在活化后48h,而Cocktail活化組峰值出現(xiàn)在活化后60h,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。Cosmc轉(zhuǎn)錄及蛋白水平、T-synthase活性隨活化時(shí)間延長(zhǎng)先降低后升高,變化趨勢(shì)與Tn抗原表達(dá)水平相反,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論 T細(xì)胞活化過(guò)程中Tn抗原表達(dá)呈動(dòng)態(tài)變化,表達(dá)水平與Cosmc轉(zhuǎn)錄及蛋白水平下降、T-synthase活性降低密切相關(guān),為以Tn抗原為靶點(diǎn)治療由T細(xì)胞活化引起的免疫性疾病提供了重要依據(jù)。
[Abstract]:Objective to investigate the relationship between the expression of Tn antigen and the transcription and protein expression of Cosmc during T cell activation. Methods 30 healthy volunteers, 15 males and 15 females, with an average age of 34 鹵8.9, were enrolled in this study. Mononuclear cells (peripheral blood mononuclear cell,PBMC) were isolated from peripheral venous blood and heparin by Ficoll density gradient centrifugation. The purified CD3 T cells were isolated by immunomagnetizing beads and stimulated by T cell activating agent Cocktail,CD3/CD28Dynabeads for 12, 24, 36, 48, 60, 72 h, respectively. The percentage of Tn cells and the average fluorescence intensity of Tn antigen in CD3 T cells were detected by flow cytometry at different time points before and after activation. RT-PCR and Western blot were used to detect the transcription and protein expression of Cosmc in T cells at different time points before and after activation. T-synthase activity was detected by fluorescence analysis and statistically analyzed by SPASS13.0 software. To elucidate the relationship between the dynamic changes of Tn antigen on activated T cells and Cosmc. Results Cocktail,CD3/CD28Dynabeads could promote the expression of Tn antigen on the surface of activated T cells, and the average fluorescence intensity of Tn antigen expression increased at first and then decreased with the prolongation of activation time. The peak value of Tn cell percentage appeared at 48 h after activation, while that of Cocktail activation group appeared at 60 h after activation, the difference was statistically significant (P 0.05). The activity of T-synthase decreased at first and then increased with the prolongation of activation time, and the change trend was opposite to the expression level of Tn antigen, and the difference was statistically significant (P 0.05). Conclusion the expression of Tn antigen changes dynamically during T cell activation, and the expression level is closely related to the decrease of Cosmc transcription and protein level and the decrease of T-synthase activity, which provides an important basis for the treatment of immune diseases caused by T cell activation with Tn antigen as a target.
【作者單位】： 濱州醫(yī)學(xué)院免疫學(xué)教研室泰山學(xué)者團(tuán)隊(duì);臨淄區(qū)人民醫(yī)院;濱州醫(yī)學(xué)院附屬醫(yī)院;
【基金】：國(guó)家自然科學(xué)基金項(xiàng)目(No.30972778) 山東省自然科學(xué)基金項(xiàng)目(No.Y2007C143,ZR2014HM020)
【分類號(hào)】：R392

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