本文選題：TGF-β_1 + Foxp3��； 參考：《華中科技大學(xué)》2007年博士論文

【摘要】： 第一部分TGF-β1對(duì)同種反應(yīng)性T細(xì)胞增殖能力及CD25表達(dá)的影響 [目的]研究TGF-β1對(duì)同種反應(yīng)性T細(xì)胞增殖能力及CD25表達(dá)水平的影響。 [方法]以絲裂霉素處理的Balb/C(H-2d)裸鼠脾細(xì)胞為刺激細(xì)胞,CFDA-SE標(biāo)記的C57BL/6(H-2b)小鼠的T細(xì)胞為反應(yīng)細(xì)胞進(jìn)行混合淋巴細(xì)胞培養(yǎng)(mixed lymphocytes culture, MLC)。分別設(shè)立對(duì)照組(TGF-β1 0ng/ml)、低濃度組(TGF-β1 1.0ng/ml)、中等濃度組(TGF-β1 5.0ng/ml)和高濃度組(TGF-β1 10.0ng/ml)。MLC結(jié)束后利用Alamar Blue法檢測(cè)T細(xì)胞增殖活性,流式細(xì)胞儀(FACS)檢測(cè)CD3、CD25表達(dá)水平。同時(shí)設(shè)立IL-2組(TGF-β1 5.0ng/ml +外源性IL-2 100u/ml),以觀測(cè)其對(duì)TGF-β1生物學(xué)效應(yīng)的影響。 [結(jié)果] TGF-β1可明顯降低同種抗原活化T細(xì)胞的增殖反應(yīng)強(qiáng)度(對(duì)照組vs低濃度組,p0.05),而且隨著TGF-β1劑量加大,免疫抑制能力增強(qiáng)(低濃度組vs中濃度組,p0.05)。TGF-β1在抑制T細(xì)胞增殖的同時(shí),也使得CD25+T細(xì)胞比例上調(diào)(對(duì)照組vs低濃度組,p0.05),但并無明顯的劑量依耐性。加入外源性IL-2后,T細(xì)胞增殖活性增強(qiáng)(IL-2組vs中濃度組,p0.05),同時(shí)CD25+T細(xì)胞比例降低(IL-2組vs中濃度組,p0.05)。 [結(jié)論] TGF-β1可明顯抑制同種抗原活化T細(xì)胞的增殖,并使CD25+T細(xì)胞表達(dá)增加,而外源性IL-2則可逆轉(zhuǎn)TGF-β1的免疫抑制功能,下調(diào)CD25+T細(xì)胞比例,說明TGF-β1的生物學(xué)效應(yīng)要受到外源性IL-2的影響。 第二部分TGF-β1誘導(dǎo)CD4+CD25-T細(xì)胞分化為CD4+CD25+調(diào)節(jié)性T細(xì)胞 [目的]研究TGF-β1誘導(dǎo)CD4+CD25+調(diào)節(jié)性T細(xì)胞分化的能力及其相關(guān)機(jī)理。 [方法] 1、利用絲裂霉素處理的Balb/C裸鼠脾細(xì)胞刺激C57BL/6小鼠T細(xì)胞,同時(shí)給予TGF-β1予以干預(yù)(按TGF-β1劑量不同設(shè)立4組:對(duì)照組,低濃度組,中濃度組和高濃度組),共同培養(yǎng)5天后,用流式細(xì)胞儀檢測(cè)CD4+CD25+T細(xì)胞比例;同時(shí)用RT-PCR檢測(cè)培養(yǎng)細(xì)胞中Foxp3的表達(dá)水平。2、在第一部分實(shí)驗(yàn)基礎(chǔ)上,通過MACS分離獲取C57BL/6小鼠CD4+CD25-T細(xì)胞,用同種抗原刺激后,給予TGF-β1干預(yù),培養(yǎng)5天后分離CD4+CD25+T細(xì)胞,利用混合淋巴細(xì)胞培養(yǎng)系統(tǒng)檢測(cè)其抑制淋巴細(xì)胞增殖的能力。[結(jié)果] T細(xì)胞經(jīng)同種抗原刺激后,在中、高濃度TGF-β1誘導(dǎo)下CD4+CD25+T細(xì)胞比例均明顯升高,Foxp3的表達(dá)水平也相應(yīng)增加,與對(duì)照組相比有顯著性差異(p0.05)。TGF-β1可直接誘導(dǎo)CD4+CD25-T細(xì)胞轉(zhuǎn)化為CD4+CD25+T細(xì)胞,轉(zhuǎn)化后的CD4+CD25+T細(xì)胞能有效抑制淋巴細(xì)胞增殖。 [結(jié)論] TGF-β1可誘導(dǎo)CD4+CD25-T細(xì)胞轉(zhuǎn)化為CD4+CD25+Treg,促進(jìn)其表達(dá)Foxp3,并能抑制淋巴細(xì)胞增殖。 第三部分TGF-β1誘導(dǎo)的調(diào)節(jié)性T細(xì)胞體內(nèi)輸注延長小鼠皮膚移植物存活 [目的]利用TGF-β1體外誘導(dǎo)na?ve T細(xì)胞分化為調(diào)節(jié)性T細(xì)胞,通過體內(nèi)輸注延長小鼠皮膚移植物存活時(shí)間,并研究其相關(guān)機(jī)理。 [方法]根據(jù)誘導(dǎo)條件不同分為三組:對(duì)照組(加入IL-2培養(yǎng)的C57BL/6小鼠T細(xì)胞)、MLR組(經(jīng)同種抗原刺激活化的C57BL/6小鼠T細(xì)胞)和TGF-β組(經(jīng)同種抗原刺激活化的C57BL/6小鼠T細(xì)胞,同時(shí)加入5.0ng/ml TGF-β1誘導(dǎo))。利用FACS檢測(cè)CD4+CD25+T細(xì)胞比例,并用RT-PCR檢測(cè)Foxp3的表達(dá)水平。建立小鼠皮膚移植模型,并于0、1、2和3d輸注上述細(xì)胞,觀察皮膚移植物存活時(shí)間。術(shù)后第9天,取部分受鼠行移植物病理檢測(cè),用FACS檢測(cè)脾臟中Th1、Th2和CD4+CD25+Treg比例,并用Alamar Blue法檢測(cè)淋巴細(xì)胞增殖能力。 [結(jié)果] TGF-β組中CD4+CD25+T細(xì)胞比例高于對(duì)照組和MLR組(p0.05),且其高表達(dá)Foxp3。將培養(yǎng)的細(xì)胞輸注給受鼠后發(fā)現(xiàn),輸注MLR組細(xì)胞的受鼠其移植物平均存活時(shí)間(mean survival time, MST)為(9.4±1.3)d,低于對(duì)照組(p0.05);而輸注TGF-β組細(xì)胞小鼠MST為(22.8±1.9)d,較對(duì)照組和MLR組明顯延長(p0.05)。病理檢測(cè)亦顯示TGF-β組受鼠移植物結(jié)構(gòu)完整,無明顯淋巴細(xì)胞浸潤。FACS結(jié)果顯示TGF-β組小鼠體內(nèi)Th1細(xì)胞(CD4+TIM-3+)比例低于對(duì)照組和MLR組(p0.05),而Th2細(xì)胞(CD4+TIM-1+)比例則與MLR組類似(p0.05),但低于對(duì)照組(p0.05);而且TGF-β組中CD4+CD25+Treg比例明顯高于對(duì)照組和MLR組(p0.05)。用Alamar Blue法檢測(cè)受鼠外周淋巴細(xì)胞增殖活性顯示,TGF-β組受鼠淋巴細(xì)胞增殖能力被明顯抑制,低于對(duì)照組(p0.05)。 [結(jié)論] TGF-β1可誘導(dǎo)T細(xì)胞分化為具有抑制能力的調(diào)節(jié)性T細(xì)胞,將誘導(dǎo)后的細(xì)胞進(jìn)行過繼輸注可使小鼠體內(nèi)CD4+CD25+Treg比例升高,同時(shí)抑制Th1和Th2細(xì)胞分化擴(kuò)增,并使得外周淋巴細(xì)胞增殖能力減弱,從而延長小鼠皮膚移植物存活時(shí)間。 第四部分調(diào)節(jié)性T細(xì)胞通過TIM-3-TIM-3L通路抑制淋巴細(xì)胞增殖 [目的]研究TIM-3-TIM-3L通路在調(diào)節(jié)性T細(xì)胞免疫抑制效應(yīng)中的作用。 [方法]用MACS從成年C57BL/6分離獲取天然調(diào)節(jié)性T細(xì)胞(natural Treg, nTreg),并利用TGF-β1誘導(dǎo)na?ve T細(xì)胞分化為誘導(dǎo)性調(diào)節(jié)性T細(xì)胞(induced Treg, iTreg),隨后通過RT-PCR檢測(cè)其表達(dá)Foxp3和TIM-3L的水平,及其對(duì)淋巴細(xì)胞增殖的抑制能力。使用TIM-3融合蛋白(TIM-3.Ig)和anti-TIM-3處理nTreg和iTreg,對(duì)TIM-3-TIM-3L通路進(jìn)行阻斷,隨后利用MLC體系檢測(cè)其免疫抑制能力的變化情況。 [結(jié)果]分離獲取的nTreg和iTreg均可表達(dá)Foxp3。TIM-3L(galectin-9)在脾細(xì)胞高表達(dá),同時(shí)在nTreg和iTreg也檢測(cè)到有g(shù)alectin-9表達(dá),但表達(dá)水平低于脾細(xì)胞(p0.05)。用TIM-3.Ig對(duì)nTreg和iTreg進(jìn)行干預(yù)后發(fā)現(xiàn),nTreg和iTreg抑制淋巴細(xì)胞增殖的能力部分減弱,與未干預(yù)組差異顯著(p0.05),但nTreg和iTreg之間無差異(p0.05);而用anti-TIM-3處理nTreg和iTreg后并未明顯減弱其抑制淋巴細(xì)胞增殖的能力(p0.05)。 [結(jié)論] TGF-β1可誘導(dǎo)iTreg產(chǎn)生。nTreg和iTreg均表達(dá)TIM-3L,并可通過TIM-3-TIM-3L通路抑制淋巴細(xì)胞增殖;但阻斷TIM-3-TIM-3L通路后Treg仍可部分抑制淋巴細(xì)胞增殖,表明TIM-3-TIM-3L通路不是Treg發(fā)揮免疫抑制效應(yīng)的唯一通路。
[Abstract]:Part one the effect of TGF- beta 1 on the proliferation and CD25 expression of allogeneic T cells
[Objective] to study the effect of TGF- beta 1 on the proliferation and CD25 expression of allogeneic T cells.
[Methods] Balb/C (H-2d) nude mice were treated with mitomycin as the stimulating cells, and the T cells of the CFDA-SE labeled C57BL/6 (H-2b) mice were used for mixed lymphocyte culture (mixed lymphocytes culture, MLC). The control group (TGF- beta 1 0ng/ml), the low concentration group (beta 1), and the medium concentration group (C57BL/6 beta 1) were set up respectively. After the end of the high concentration group (TGF- beta 1 10.0ng/ml).MLC, the proliferation activity of T cells was detected by Alamar Blue, and the level of CD3 and CD25 expression was detected by flow cytometry (FACS), and the IL-2 group (TGF- beta 1 5.0ng/ml + exogenous hormones) was set up to observe the effect of its biological effect on the beta 1.
[results] TGF- beta 1 could significantly reduce the proliferation reaction intensity of the alloantigen activated T cells (the control group, vs low concentration group, P0.05), and with the increase of TGF- beta 1, the immunosuppressive ability enhanced (the concentration group of vs in the low concentration group, P0.05).TGF- beta 1, while inhibiting the T cell proliferation, also increased the ratio of CD25+T cells (vs low concentration group of control group, P0). .05), but there was no obvious dose dependent. After adding exogenous IL-2, the proliferation activity of T cells was enhanced (the concentration group of vs in group IL-2, P0.05), and the proportion of CD25+T cells decreased (the concentration group in IL-2 group vs, P0.05).
[Conclusion] TGF- beta 1 can inhibit the proliferation of T cells and increase the expression of CD25+T cells, while exogenous IL-2 can reverse the immunosuppressive function of TGF- beta 1 and reduce the proportion of CD25+T cells, indicating that the biological effect of TGF- beta 1 is affected by exogenous IL-2.
The second part, TGF- beta 1, induces CD4+CD25-T cells to differentiate into CD4+CD25+ regulatory T cells.
[Objective] to investigate the ability of TGF- beta 1 to induce differentiation of CD4+CD25+ regulatory T cells and its related mechanisms.
[method] 1, Balb/C nude mouse splenocytes treated with mitomycin were used to stimulate T cells in C57BL/6 mice, and TGF- beta 1 was given at the same time. 4 groups were set up according to the dosage of TGF- beta 1: the control group, the low concentration group, the medium concentration group and the high concentration group. The proportion of CD4+CD25+T cells was detected by the flow cytometry for 5 days, and the culture was detected by RT-PCR. The expression level of Foxp3 in the cells was.2. On the basis of the first part of the experiment, the CD4+CD25-T cells of C57BL/6 mice were obtained by MACS separation. After the stimulation of the same antigen, TGF- beta 1 was given, and CD4+CD25+T cells were isolated after 5 days, and the ability to inhibit the proliferation of lymphocytes was detected by mixed lymphocyte culture system. [results] T cells were the same. After the stimulation of the antigen, the proportion of CD4+CD25+T cells induced by high concentration of TGF- beta 1 increased obviously, and the expression level of Foxp3 increased correspondingly. Compared with the control group, there was a significant difference (P0.05).TGF- beta 1 could directly induce the transformation of CD4+CD25-T cells into CD4+CD25+T cells, and the transformed CD4+CD25+T cells could effectively inhibit the proliferation of lymphocytes.
[Conclusion] TGF- beta 1 can induce CD4+CD25-T cells to transform into CD4+CD25+Treg, promote its expression of Foxp3, and inhibit lymphocyte proliferation.
The third part is the prolongation of mouse skin graft survival by in vivo infusion of TGF- beta 1 regulatory T cells.
[Objective] to induce na? Ve T cells to differentiate into regulatory T cells by using TGF- beta 1 in vitro, and to prolong the survival time of mouse skin graft by infusion in vivo, and to study its mechanism.
[Methods] according to the different induction conditions, the control group was divided into three groups: the control group (C57BL/6 mouse T cells cultured with IL-2), the MLR group (C57BL/6 mice T cells activated by the same antigen stimulation) and the TGF- beta group (T cells activated by the alloantigen stimulated C57BL/6 mice, and 5.0ng/ml TGF- beta 1 induced by 5.0ng/ml). The expression level of Foxp3 was detected by RT-PCR. The mice skin transplantation model was established and the cells were transfused with 0,1,2 and 3D to observe the survival time of the skin graft. Ninth days after the operation, the pathological examination of the graft was taken, the proportion of Th1, Th2 and CD4+CD25+Treg in the spleen was detected by FACS, and the lymphocyte proliferation was detected by Alamar Blue.
[results] the proportion of CD4+CD25+T cells in the TGF- beta group was higher than that of the control group and the MLR group (P0.05), and the high expression of Foxp3. showed that the cells of the cultured cells were transfused to the rats. The average survival time (mean survival time, MST) of the transfused MLR group was (9.4 + 1.3) d, lower than the control group (P0.05). (22. 8 + 1.9) d, compared with the control group and MLR group (P0.05), the pathological examination also showed that the structure of the TGF- beta group was complete, and no obvious lymphocyte infiltration.FACS results showed that the proportion of Th1 cells (CD4+TIM-3+) in the TGF- beta group was lower than that of the control group and the MLR group (P0.05), but the proportion of Th2 cells (CD4+TIM-1+) was similar to that of the group, but lower than that of the group. In the control group (P0.05), the proportion of CD4+CD25+Treg in the TGF- beta group was significantly higher than that in the control group and the MLR group (P0.05). The proliferation activity of the peripheral lymphocyte in the rats was detected by Alamar Blue method, and the proliferation ability of the lymphocytes in the TGF- beta group was significantly inhibited, lower than that of the control group (P0.05).
[Conclusion] TGF- beta 1 can induce T cells to differentiate into regulatory T cells with inhibitory ability. The adoptive infusion of induced cells can increase the proportion of CD4+CD25+Treg in mice, inhibit the differentiation and amplification of Th1 and Th2 cells, and reduce the proliferation of peripheral lymphocytes, thus prolonging the survival time of the skin graft in mice.
The fourth part of regulatory T cells inhibit lymphocyte proliferation through TIM-3-TIM-3L pathway.
[Objective] to study the role of TIM-3-TIM-3L pathway in regulating the immunosuppressive effect of regulatory T cells.
[Methods] natural regulatory T cells (NATURAL Treg, nTreg) were isolated from adult C57BL/6, and na? Ve T cells were induced by TGF- beta 1 to differentiate into inducible T cells (induced). TIM-3.Ig (anti-TIM-3) and nTreg (nTreg) and iTreg were used to block the TIM-3-TIM-3L pathway, and then the MLC system was used to detect the changes of the immunosuppressive ability.
[results] the isolated nTreg and iTreg could express the high expression of Foxp3.TIM-3L (galectin-9) in the spleen cells, while the expression of galectin-9 was also detected in nTreg and iTreg, but the expression level was lower than that of the spleen cells (P0.05). There was significant difference (P0.05) in the untreated group, but there was no difference between nTreg and iTreg (P0.05), while nTreg and iTreg treated with anti-TIM-3 did not significantly weaken the ability to inhibit lymphocyte proliferation (P0.05).
[Conclusion] TGF- beta 1 can induce iTreg to produce.NTreg and iTreg to express TIM-3L, and can inhibit lymphocyte proliferation through TIM-3-TIM-3L pathway, but Treg still partially inhibits lymphocyte proliferation after blocking TIM-3-TIM-3L pathway, indicating that TIM-3-TIM-3L pathway is not the only pathway for Treg to play an immunosuppressive response.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392

【共引文獻(xiàn)】

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本文編號(hào)：2004261