调节性T细胞在糖尿病患者外周血的表达及其体外扩增模型的建立

发布时间：2018-06-24 01:25

本文选题：调节性T淋巴细胞 + 糖尿病　；参考：《南京医科大学》2012年硕士论文

【摘要】：目的探讨不同类型糖尿病患者外周血FOXP3+调节性T细胞数量的差异及其临床意义。方法研究分为1型糖尿病组（n=43例），2型糖尿病组（n=16例）和健康对照组（n=19例）。采用放射配体法检测胰岛自身抗体ZnT8A、GADA和ICA。采用细胞膜打孔和三色荧光标记流式细胞术检测外周血CD4~+T的细胞群中CD4~+CD25~+FOXP3+T细胞所占百分比、CD4~+CD25~+T细胞所占百分比。结果①1型糖尿病患者和2型糖尿病患者的外周血中CD4~+CD25~+T细胞占CD4~+T淋巴细胞的百分比均低于健康志愿者，差异有统计学意义（P0.05）。②1型糖尿病患者的外周血中CD4~+CD25~+FOXP3+T细胞占CD4~+T淋巴细胞的百分比低于健康志愿者和2型糖尿病患者，差异有统计学意义（P0.05）。③1型糖尿病患者的病程与外周血CD4~+CD25~+FOXP3+T细胞数量、CD4~+CD25~+T细胞数量均无相关性。结论1型糖尿病患者外周血FOXP3+调节性T细胞数量下降，FOXP3+调节性T细胞数量的下降程度与病程无关。2型糖尿病患者外周血FOXP3+调节性T细胞数量无异常。FOXP3+是调节性T细胞的特征性标志。目的建立人外周血CD4~+CD25~+CD127~(dim/-)TT细胞体外分选技术，探讨分选的CD4~+CD25~+CD127~(dim/-)TT细胞用于调节性T细胞体外扩增的可行性。方法采用Ficoll密度梯度离心法从1U粒细胞中获取外周血单核淋巴细胞(Peripheral blood mononuclear cells, PBMC)。利用免疫磁珠分选法从PBMC中分选CD4~+CD25~+CD127~(dim/-)TT细胞。采用细胞膜打孔和三色荧光标记流式细胞术检测分选的细胞表面标志和FOXP3阳性率。采用RT-PCR检测CD4~+CD25~+CD127~(dim/-)TT细胞内FOXP3mRNA表达量。结果①经免疫磁珠法分选的CD4~+CD25~+CD127~(dim/-)TT细胞纯度为（87.4士2.6）%，其中FOXP3阳性率（85.6士4.2）%；②CD4~+CD25~+CD127~(dim/-)TT细胞内FOXP3mRNA表达水平显著高于PBMC和CD4-CD127highT细胞，差异有统计学意义（P0.05）。结论免疫磁珠两步法可以高效的从外周血中分选出CD4~+CD25~+CD127~(dim/-)TT细胞。CD4~+CD25~+CD127~(dim/-)TT细胞群高表达FOXP3，，是体外扩增Tregs的可靠细胞来源。目的建立CD4~+CD25~+CD127~(dim/-)TT细胞体外扩增体系，探讨影响CD4~+CD25~+CD127~(dim/-)TT细胞体外扩增效率和纯度的相关因素。方法将分选获得的CD4~+CD25~+CD127~(dim/-)TT细胞按104细胞/100μL/孔接种到96孔板，给予anti-CD3/anti-CD28磁珠刺激，根据不同培养条件加入IL-2、和/或rapamycin，于不同的体外扩增周期收获CD4~+CD25~+CD127~(dim/-)TT细胞。采用Annexin V和PI荧光标记流式细胞术检测扩增后获得的Tregs的调亡率。采用细胞膜打孔和三色荧光标记流式细胞术检测扩增后获得的Tregs的纯度。采用混合淋巴细胞培养检测扩增后Tregs免疫抑制功能。结果①磁珠分选后，使用anti-CD3/anti-CD28磁珠联合1000U/ml IL-2刺激CD4~+CD25~+CD127~(dim/-)TT细胞增殖，Tregs数目在培养第1周扩增（67±5）倍，第2周扩增（20±3）倍。②经过第1周的体外扩增后，Tregs的调亡率为（14.2±2.1）%，其中（10.5±1.2）%为晚期凋亡；经过第2周的体外扩增后，T细胞的调亡率为（16.6±3.2）%，其中（10.6±2.3）%为晚期凋亡；经过第3周的体外扩增后，Tregs的调亡率为27.4%，其中26.9%为晚期凋亡。③经过2周的体外扩增， Tregs纯度由（87.4士2.6）%下降到（65.1±4.0）%，但是CD4~+CD25~+T细胞比例仍接近90%。④混合淋巴细胞培养实验显示体外扩增的Tregs对同种异体效应T细胞的增殖有明显的抑制作用。结论qanti-CD3/anti-CD28磁珠联合1000U/ml的IL-2可以在体外高效扩增人外周血Tregs，合适的扩增时间是2周。扩增的Tregs仍具有免疫抑制性。成功建立了CD4~+CD25~+CD127~(dim/-)TT细胞体外扩增体系，为Tregs的临床应用奠定了基础。
[Abstract]:Objective to investigate the difference of the number of FOXP3+ regulatory T cells in peripheral blood of patients with different types of diabetes and its clinical significance.
Methods the study was divided into type 1 diabetes group (n=43), type 2 diabetes group (n=16) and healthy control group (n=19). The radioligand method was used to detect the islet autoantibody ZnT8A, GADA and ICA. were detected by cell membrane perforation and tricolor fluorescence labeling flow cytometry to determine the percentage of CD4~+CD25~+FOXP3+T cells in the cell group of peripheral blood CD4~+T, CD4~+CD The percentage of 25~+T cells.
Results (1) the percentage of CD4~+CD25~+T cells in peripheral blood of type 1 diabetes and type 2 diabetic patients was lower than that of healthy volunteers. The difference was statistically significant (P0.05). (2) the percentage of CD4~+CD25~+FOXP3+T cells in peripheral blood of type 1 diabetes patients was lower than that of healthy volunteers and type 2 sugar. The difference was statistically significant (P0.05). (3) there was no correlation between the duration of the patients with type 1 diabetes and the number of CD4~+CD25~+FOXP3+T cells in peripheral blood and the number of CD4~+CD25~+T cells.
Conclusion the number of FOXP3+ regulatory T cells in peripheral blood decreased in patients with type 1 diabetes, the decrease in the number of FOXP3+ regulatory T cells and the course of disease were not related to the number of FOXP3+ regulated T cells in peripheral blood of patients with.2 type diabetes, which was the characteristic marker of regulatory T cells.
Objective to establish the in vitro separation technique of human peripheral blood CD4~+CD25~+CD127~ (dim/-) TT cells in vitro, and to explore the feasibility of the selected CD4~+CD25~+CD127~ (dim/-) TT cells for the amplification of regulatory T cells in vitro.
Methods Ficoll density gradient centrifugation was used to obtain peripheral blood mononuclear lymphocytes (Peripheral blood mononuclear cells, PBMC) from 1U granulocytes. CD4~+CD25~+CD127~ (dim/-) TT cells were selected from PBMC by immunomagnetic beads. Cell surface markers were detected by cell membrane perforation and three color fluorescence labeling flow cytometry. And FOXP3 positive rate. RT-PCR was used to detect FOXP3mRNA expression in CD4~+CD25~+CD127~ (dim/-) TT cells.
Results (1) the purity of CD4~+CD25~+CD127~ (dim/-) TT cells by immunomagnetic beads was (87.4)% (2.6)%, and the positive rate of FOXP3 (85.6. 4.2)%, and the level of FOXP3mRNA expression in CD4~+CD25~+CD127~ (dim/-) TT cells was significantly higher than that of PBMC and CD4-CD127highT cells, and the difference was statistically significant (P0.05).
Conclusion the two step method of immunomagnetic beads can be used to efficiently select the.CD4~+CD25~+CD127~ (dim/-) TT cell group of CD4~+CD25~+CD127~ (dim/-) TT cells from peripheral blood to express FOXP3, which is a reliable cell source for the amplification of Tregs in vitro.
Objective to establish an in vitro amplification system of CD4~+CD25~+CD127~ (dim/-) TT cells and to explore the factors affecting the amplification efficiency and purity of CD4~+CD25~+CD127~ (dim/-) TT cells in vitro.
Methods the selected CD4~+CD25~+CD127~ (dim/-) TT cells were inoculated to 96 orifice plates by 104 cell /100 mu L/ holes and stimulated by anti-CD3/anti-CD28 magnetic beads. IL-2, and / or rapamycin were added according to the different culture conditions, and CD4~+CD25~+CD127~ (dim/-) TT cells were harvested in different extracorporeal cycles. The apoptosis rate of Tregs obtained after amplification was detected. The purity of Tregs was detected by cell membrane perforation and three color fluorescence labeling flow cytometry. The immune inhibitory function of Tregs was detected by mixed lymphocyte culture.
Results (1) after the magnetic beads were selected, the proliferation of CD4~+CD25~+CD127~ (dim/-) TT cells was stimulated by anti-CD3/anti-CD28 magnetic beads combined with 1000U/ml IL-2. The number of Tregs was amplified (67 + 5) times (20 + 3) times at second weeks (20 + 3) times. After first weeks of expansion, the rate of Tregs modulation was (14.2 + 2.1)%, and (10.5 + 67)% was late apoptosis. After second weeks in vitro amplification, the apoptosis rate of T cells was (16.6 + 3.2)%, of which (10.6 + 2.3)% was late apoptosis. After third weeks of expansion in vitro, the rate of Tregs was 27.4%, and 26.9% was late apoptosis. (3) after 2 weeks in vitro amplification, the purity of Tregs decreased from (87.4 2.6)% to (65.1 + 4)%, but the proportion of CD4~+CD25~+T cells was still connected. Near 90%. 4 mixed lymphocyte culture experiments showed that Tregs amplification in vitro significantly inhibited the proliferation of T cells.
Conclusion qanti-CD3/anti-CD28 magnetic beads combined with 1000U/ml IL-2 can effectively amplify human peripheral blood Tregs in vitro. The appropriate amplification time is 2 weeks. The amplified Tregs is still immunosuppressive. The amplification system of CD4~+CD25~+CD127~ (dim/-) TT cells in vitro has been successfully established, which lays a foundation for the application of Tregs in bed.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R587.1;R-332

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