本文選題：調(diào)節(jié)性T淋巴細(xì)胞 + 糖尿病　； 參考：《南京醫(yī)科大學(xué)》2012年碩士論文

【摘要】：目的探討不同類型糖尿病患者外周血FOXP3+調(diào)節(jié)性T細(xì)胞數(shù)量的差異及其臨床意義。 方法研究分為1型糖尿病組（n=43例），2型糖尿病組（n=16例）和健康對(duì)照組（n=19例）。采用放射配體法檢測(cè)胰島自身抗體ZnT8A、GADA和ICA。采用細(xì)胞膜打孔和三色熒光標(biāo)記流式細(xì)胞術(shù)檢測(cè)外周血CD4~+T的細(xì)胞群中CD4~+CD25~+FOXP3+T細(xì)胞所占百分比、CD4~+CD25~+T細(xì)胞所占百分比。 結(jié)果①1型糖尿病患者和2型糖尿病患者的外周血中CD4~+CD25~+T細(xì)胞占CD4~+T淋巴細(xì)胞的百分比均低于健康志愿者，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。②1型糖尿病患者的外周血中CD4~+CD25~+FOXP3+T細(xì)胞占CD4~+T淋巴細(xì)胞的百分比低于健康志愿者和2型糖尿病患者，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。③1型糖尿病患者的病程與外周血CD4~+CD25~+FOXP3+T細(xì)胞數(shù)量、CD4~+CD25~+T細(xì)胞數(shù)量均無(wú)相關(guān)性。 結(jié)論1型糖尿病患者外周血FOXP3+調(diào)節(jié)性T細(xì)胞數(shù)量下降，F(xiàn)OXP3+調(diào)節(jié)性T細(xì)胞數(shù)量的下降程度與病程無(wú)關(guān)。2型糖尿病患者外周血FOXP3+調(diào)節(jié)性T細(xì)胞數(shù)量無(wú)異常。FOXP3+是調(diào)節(jié)性T細(xì)胞的特征性標(biāo)志。 目的建立人外周血CD4~+CD25~+CD127~(dim/-)TT細(xì)胞體外分選技術(shù)，探討分選的CD4~+CD25~+CD127~(dim/-)TT細(xì)胞用于調(diào)節(jié)性T細(xì)胞體外擴(kuò)增的可行性。 方法采用Ficoll密度梯度離心法從1U粒細(xì)胞中獲取外周血單核淋巴細(xì)胞(Peripheral blood mononuclear cells, PBMC)。利用免疫磁珠分選法從PBMC中分選CD4~+CD25~+CD127~(dim/-)TT細(xì)胞。采用細(xì)胞膜打孔和三色熒光標(biāo)記流式細(xì)胞術(shù)檢測(cè)分選的細(xì)胞表面標(biāo)志和FOXP3陽(yáng)性率。采用RT-PCR檢測(cè)CD4~+CD25~+CD127~(dim/-)TT細(xì)胞內(nèi)FOXP3mRNA表達(dá)量。 結(jié)果①經(jīng)免疫磁珠法分選的CD4~+CD25~+CD127~(dim/-)TT細(xì)胞純度為（87.4士2.6）%，其中FOXP3陽(yáng)性率（85.6士4.2）%；②CD4~+CD25~+CD127~(dim/-)TT細(xì)胞內(nèi)FOXP3mRNA表達(dá)水平顯著高于PBMC和CD4-CD127highT細(xì)胞，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。 結(jié)論免疫磁珠兩步法可以高效的從外周血中分選出CD4~+CD25~+CD127~(dim/-)TT細(xì)胞。CD4~+CD25~+CD127~(dim/-)TT細(xì)胞群高表達(dá)FOXP3，，是體外擴(kuò)增Tregs的可靠細(xì)胞來(lái)源。 目的建立CD4~+CD25~+CD127~(dim/-)TT細(xì)胞體外擴(kuò)增體系，探討影響CD4~+CD25~+CD127~(dim/-)TT細(xì)胞體外擴(kuò)增效率和純度的相關(guān)因素。 方法將分選獲得的CD4~+CD25~+CD127~(dim/-)TT細(xì)胞按104細(xì)胞/100μL/孔接種到96孔板，給予anti-CD3/anti-CD28磁珠刺激，根據(jù)不同培養(yǎng)條件加入IL-2、和/或rapamycin，于不同的體外擴(kuò)增周期收獲CD4~+CD25~+CD127~(dim/-)TT細(xì)胞。采用Annexin V和PI熒光標(biāo)記流式細(xì)胞術(shù)檢測(cè)擴(kuò)增后獲得的Tregs的調(diào)亡率。采用細(xì)胞膜打孔和三色熒光標(biāo)記流式細(xì)胞術(shù)檢測(cè)擴(kuò)增后獲得的Tregs的純度。采用混合淋巴細(xì)胞培養(yǎng)檢測(cè)擴(kuò)增后Tregs免疫抑制功能。 結(jié)果①磁珠分選后，使用anti-CD3/anti-CD28磁珠聯(lián)合1000U/ml IL-2刺激CD4~+CD25~+CD127~(dim/-)TT細(xì)胞增殖，Tregs數(shù)目在培養(yǎng)第1周擴(kuò)增（67±5）倍，第2周擴(kuò)增（20±3）倍。②經(jīng)過(guò)第1周的體外擴(kuò)增后，Tregs的調(diào)亡率為（14.2±2.1）%，其中（10.5±1.2）%為晚期凋亡；經(jīng)過(guò)第2周的體外擴(kuò)增后，T細(xì)胞的調(diào)亡率為（16.6±3.2）%，其中（10.6±2.3）%為晚期凋亡；經(jīng)過(guò)第3周的體外擴(kuò)增后，Tregs的調(diào)亡率為27.4%，其中26.9%為晚期凋亡。③經(jīng)過(guò)2周的體外擴(kuò)增， Tregs純度由（87.4士2.6）%下降到（65.1±4.0）%，但是CD4~+CD25~+T細(xì)胞比例仍接近90%。④混合淋巴細(xì)胞培養(yǎng)實(shí)驗(yàn)顯示體外擴(kuò)增的Tregs對(duì)同種異體效應(yīng)T細(xì)胞的增殖有明顯的抑制作用。 結(jié)論qanti-CD3/anti-CD28磁珠聯(lián)合1000U/ml的IL-2可以在體外高效擴(kuò)增人外周血Tregs，合適的擴(kuò)增時(shí)間是2周。擴(kuò)增的Tregs仍具有免疫抑制性。成功建立了CD4~+CD25~+CD127~(dim/-)TT細(xì)胞體外擴(kuò)增體系，為T(mén)regs的臨床應(yīng)用奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R587.1;R-332

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