本文關(guān)鍵詞：IL-2上調(diào)淋巴細(xì)胞SOCS-3表達(dá)阻滯Th1分化抑制小鼠急性GVHD的實(shí)驗(yàn)研究　出處：《復(fù)旦大學(xué)》2007年博士論文　論文類型：學(xué)位論文

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【摘要】： 目的:研究小劑量IL-2預(yù)孵育移植物后對(duì)小鼠急性GVHD的影響,探討小劑量IL-2預(yù)孵育后同種異基因淋巴細(xì)胞中SOCS-3蛋白表達(dá)的變化在此過(guò)程中的作用與機(jī)制。 方法: 1、用IL-2預(yù)孵育DO11.10轉(zhuǎn)基因小鼠naive T淋巴細(xì)胞、C57BL/6N小鼠脾細(xì)胞和C57BL/6N小鼠na(?)veCD4+T細(xì)胞,檢測(cè)SOCS-3基因的表達(dá)變化。用OVA_(323-329)特異性抗原刺激活化DO11.10TCR轉(zhuǎn)基因小鼠T細(xì)胞,檢測(cè)T淋巴細(xì)胞經(jīng)抗原活化后SOCS-3基因的表達(dá)變化。 2、構(gòu)建pLX-SOCS3-SN載體,建立高表達(dá)SOCS-3基因的DO11.10細(xì)胞(DS細(xì)胞),用OVA_(323-329)特異性抗原刺激活化DS細(xì)胞,檢測(cè)DS細(xì)胞的免疫應(yīng)答能力。 3、用IL-2(50U/ml)預(yù)孵育DO11.10 TCR轉(zhuǎn)基因小鼠na(?)ve T淋巴細(xì)胞、C57BL/6N小鼠脾細(xì)胞和C57BL/6N小鼠na(?)ve CD4+T細(xì)胞,然后用OVA_(323-329)特異性抗原和BALB/C小鼠異基因抗原分別對(duì)其進(jìn)行刺激,測(cè)定DO11.10 TCR轉(zhuǎn)基因小鼠na(?)ve T淋巴細(xì)胞、C57BL/6N小鼠脾細(xì)胞和C57BL/6N小鼠na(?)ve CD4+T淋巴細(xì)胞免疫增殖能力。 4、以C57BL/6N小鼠的T淋巴細(xì)胞為反應(yīng)細(xì)胞,以BALB/C小鼠滅活的脾細(xì)胞為刺激細(xì)胞。將反應(yīng)細(xì)胞在終濃度為50U/ml的IL-2中預(yù)孵育4小時(shí),然后與刺激細(xì)胞在細(xì)胞因子IL-12或者IL-4存在的情況下進(jìn)行混合淋巴細(xì)胞反應(yīng)。與對(duì)照組(即反應(yīng)細(xì)胞不給予IL-2預(yù)孵育4小時(shí))比較第14天時(shí)IL-12R_(β1)、IL-12R_(β2)和胞內(nèi)IFN-γ和IL-4的表達(dá)的差異,進(jìn)行統(tǒng)計(jì)學(xué)檢驗(yàn)。 5、受鼠為雌性BALB/C,預(yù)處理為~(137)Se TBI 5GY,分為A、B、C、D、E共5組(n=12),分別過(guò)繼性植入雄性C57BL/6N脾淋巴細(xì)胞5×10~7、1××10~7和雄性BALB/C×C57BL/6N→F1脾淋巴細(xì)胞0.5×10~7、1×10~7、5×10~7(個(gè)/只)。對(duì)存活時(shí)間超過(guò)21天的受鼠在不同時(shí)間點(diǎn)用PCR檢測(cè)供者淋巴細(xì)胞嵌合體,觀察受鼠生存時(shí)間和急性GVHD的癥狀。 6、受鼠為雌性BALB/C,預(yù)處理為~(137)Se TBI 5GY,供鼠為雄性C57BL/6N。每只受鼠均腹腔注射供鼠脾淋巴細(xì)胞3×10~7個(gè),受鼠分為A(直接植入供鼠淋巴細(xì)胞)、B(植入的淋巴細(xì)胞經(jīng)過(guò)終濃度為50 U/ml的IL-2預(yù)孵育4小時(shí))、C(植入的淋巴細(xì)胞先與滅活的BALB/C脾淋巴細(xì)胞反應(yīng)72小時(shí)再植入)、D (植入的淋巴細(xì)胞先經(jīng)過(guò)終濃度為50 U/ml的IL-2預(yù)孵育4小時(shí),然后與滅活的BALB/C脾淋巴細(xì)胞混合淋巴細(xì)胞反應(yīng)72小時(shí)再植入)共4組(n=9)。觀察期為60天,觀察受鼠生存時(shí)間和急性GVHD的癥狀,死亡受鼠均行肝臟和小腸的病理檢查了解急性GVHD的病理改變情況。觀察期結(jié)束時(shí)取每只小鼠脾細(xì)胞檢測(cè)嵌合體,取肝臟和小腸行病理檢查,了解急性GVHD的病理改變。 結(jié)果: 1、DO11.10 TCR轉(zhuǎn)基因小鼠na(?)ve T淋巴細(xì)胞、C57BL/6N小鼠脾細(xì)胞和C57BL/6小鼠na(?)ve CD4+T淋巴細(xì)胞經(jīng)IL-2預(yù)孵育后,SOCS-3的表達(dá)明顯升高,在4-6小時(shí)達(dá)到高峰。DO11.10 TCR轉(zhuǎn)基因小鼠T細(xì)胞經(jīng)OVA_(323-329)特異性抗原刺激后SOCS-3的表達(dá)明顯降低,以2-3天最低。 2、高表達(dá)SOCS-3的DS細(xì)胞對(duì)OVA_(323-329)特異性抗原免疫應(yīng)答能力明顯減弱。 3、DO11.10轉(zhuǎn)基因小鼠na(?)ve T淋巴細(xì)胞、C57BL/6N小鼠脾細(xì)胞和C57BL/6N小鼠na(?)ve CD4+T淋巴細(xì)胞經(jīng)IL-2預(yù)孵育后對(duì)OVA_(323-329)特異性抗原和同種異基因抗原免疫增殖能力明顯減弱。 4、經(jīng)IL-2預(yù)孵育后的CD4+T淋巴細(xì)胞,接受異基因抗原刺激的同時(shí)加入外源性IL-12培養(yǎng),第14天時(shí),IL-12R_(β1)、IL-12R_(β2)的表達(dá)雖然呈陽(yáng)性,但是明顯低于未經(jīng)預(yù)孵育組(P值0.05),IFN-γ與IL-4陽(yáng)性率的比值也低于未經(jīng)預(yù)孵育組(P0.05)。加入外源性IL-4培養(yǎng)時(shí),經(jīng)IL-2預(yù)孵育組IL-12R_(β1)、IL-12R_(β2)的表達(dá)也明顯明低于未經(jīng)預(yù)孵育組(P0.05),IL-4的表達(dá)明顯要高于未經(jīng)預(yù)孵育組(P0.05)。 5、TBI為5GY的免疫抑制后,在受鼠保留自身造血的情況下,過(guò)繼性同種異基因淋巴細(xì)胞可以植活。MHC分子半相合的植活率(D組)比完全不相合的(B組)高,植活時(shí)間長(zhǎng)(P0.01);在半相合的植入模型中植入的細(xì)胞量越多,植活率越高,植活時(shí)間逐漸延長(zhǎng)(P0.05)。同時(shí)仍有急性GVHD發(fā)生,MHC完全不相合(A組)移植小鼠可發(fā)生致死性急性GVHD,半相合(E組)急性GVHD明顯減輕(P0.01)。 6、IL-2(50 U/ml)預(yù)孵育同種異基因淋巴細(xì)胞4小時(shí)后,然后與滅活后的BALB/C脾淋巴細(xì)胞混合反應(yīng)72小時(shí),再過(guò)繼性植入后其急性GVHD的發(fā)生強(qiáng)度明顯減輕。 結(jié)論: 小劑量的IL-2預(yù)孵育同種異基因的淋巴細(xì)胞后,可以明顯減輕過(guò)繼性植入后急性GVHD的發(fā)生。其機(jī)制與小劑量IL-2預(yù)孵育異基因淋巴細(xì)胞上調(diào)SOCS-3的表達(dá)后,抑制其增殖反應(yīng)和阻滯Th0細(xì)胞接觸同種抗原后向Th1方向的分化有關(guān)。
[Abstract]:Objective: To study the effect of low dose IL-2 on acute GVHD in mice after preincubation with grafts, and to explore the role of SOCS-3 protein expression in allogeneic lymphocytes after low dose IL-2 preincubation.
Method:
1, with the IL-2 pre incubation of naive T lymphocytes in DO11.10 transgenic mice, C57BL/6N mice spleen cells and C57BL/6N mice Na (?) veCD4+T cells, expression of SOCS-3 gene was detected by OVA_ (323-329). The specificity of antigen activated T cells in DO11.10TCR transgenic mice, the expression detection of T lymphocyte activation antigen SOCS-3 gene.
2, we constructed pLX-SOCS3-SN vector, established DO11.10 cells (DS cells) with high expression of SOCS-3 gene, stimulated DS cells with OVA_ (323-329) specific antigen, and detected the immune response ability of DS cells.
3, IL-2 (50U/ml) TCR transgenic mice were preincubated with DO11.10 Na (?) ve T lymphocytes, spleen cells from C57BL/6N mice and C57BL/6N mice (Na? VE) CD4+T cells, followed by OVA_ (323-329) specific antigen and BALB/C mouse allogeneic antigen respectively on the stimulation, the determination of DO11.10 TCR transgenic mice Na (?) ve T lymphocytes, spleen cells from C57BL/6N mice and C57BL/6N mice (Na? VE) CD4+T lymphocyte proliferation.
4, in C57BL/6N mice T lymphocytes in BALB/C mice, inactivated spleen cells as stimulator cells. The cellular response at the final concentration of 50U/ml IL-2 in pre incubated for 4 hours, mixed lymphocyte reaction and then the existence and stimulation of cell cytokines in IL-12 or IL-4 case and control. Group (i.e. reaction cells do not give IL-2 pre incubation for 4 hours) compared to the fourteenth day IL-12R_ (beta 1), IL-12R_ (beta 2) and the differential expression of IFN- gamma and intracellular IL-4, were statistically tested.
5 recipients were female BALB/C, pretreatment of ~ (137) Se TBI 5GY, divided into A, B, C, D, E 5 group (n=12), respectively. The adoptive transfer of spleen lymphocytes of 5 male C57BL/6N * 10~7,1 * 10~7 * BALB/C * C57BL/6N, and the male F1 of spleen lymphocytes of 0.5 * 10~7,1 * 10~7,5 * 10~7 (a). The survival time in more than 21 days at different time points by PCR detection of donor lymphocyte chimerism, observe the survival time of rats and acute GVHD symptoms.
6 recipients were female BALB/C, pretreatment of ~ (137) Se TBI 5GY, C57BL/6N. for male rats each rats were injected for rat spleen lymphocytes of 3 * 10~7, the rats were divided into A (direct implantation of donor lymphocytes (B), implantation of lymphocytes after a final concentration of IL-2 after 50 U/ml incubation for 4 hours), C (the first BALB/C lymphocytes and implanted splenic lymphocyte reaction inactivated again in 72 hours, D (implant) implantation of lymphocytes after the final concentration of IL-2 were incubated with 50 U/ml incubation for 4 hours, then BALB/C and spleen lymphocyte mixed lymphocyte reaction inactivated 72 hours before implantation a total of 4) group (n=9). The observation period was 60 days, observe the survival time of rats and the acute symptoms of GVHD rats were performed by pathological examination of death in the liver and small intestine pathological changes of acute GVHD. At the end of the observation period and spleen cells from each mouse chimera detection, liver and small intestine biopsy check To find out the pathological changes of acute GVHD.
Result:
1 DO11.10 TCR Na transgenic mice (?) ve T lymphocytes, spleen cells from C57BL/6N mice and C57BL/6 mice (Na? VE) CD4+T lymphocytes after IL-2 pre incubation, the expression of SOCS-3 was significantly increased, peaked at 4-6 h.DO11.10 TCR transgenic mice T cells by OVA_ (323-329) specific antigen stimulation SOCS-3 the expression decreased obviously, with the lowest on the 2-3 day.
2, the immune response of DS cells with high expression of SOCS-3 to OVA_ (323-329) specific antigen was significantly weakened.
3, DO11.10 transgenic mice Na (VE) T lymphocytes, C57BL/6N mice spleen cells and C57BL/6N mice Na (VE) CD4+T lymphocytes after IL-2 preincubation, the proliferation of OVA_ (323-329) specific antigen and allogeneic antigen decreased significantly.
4, by IL-2 after preincubation with CD4+T lymphocytes stimulated allogeneic antigen and exogenous IL-12 cultured for fourteenth days, IL-12R_ (beta 1), IL-12R_ (beta 2) though the expression was positive, but was significantly lower than that without preincubation group (P = 0.05), the ratio of IFN- gamma and the positive rate of IL-4 is lower than that without preincubation group (P0.05). The addition of exogenous IL-4 when cultured by IL-2 preincubation group IL-12R_ (beta 1), IL-12R_ (beta 2) expression was significantly lower than that without preincubation group (P0.05), the expression of IL-4 was significantly higher than before after preincubation group (P0.05).
5, TBI 5GY in rats after immunosuppression, retain their hematopoietic condition, adoptive allogeneic lymphocyte engraftment rate can engraftment of.MHC molecular haploidentical (D group) than completely mismatched (B group), and live a long time (P0.01); more cells implantation in the model of haploidentical implantation in the graft survival rate is high, engraftment time gradually increased (P0.05). At the same time there are still acute GVHD, MHC completely mismatched (group A) transplantation mice fatal acute GVHD haploidentical (E group) with acute GVHD significantly reduced (P0.01).
6, IL-2 (50 U/ml) preincubated allogeneic lymphocytes for 4 hours, then mixed with inactivated BALB/C splenic lymphocytes for 72 hours, then the incidence of acute GVHD decreased significantly after adoptive implantation.
Conclusion:
IL-2 were incubated with small doses of sterile allogeneic lymphocytes after adoptive transfer can significantly reduce the incidence of GVHD after acute. The expression mechanism of low-dose IL-2 with pre incubation of allogeneic lymphocytes by SOCS-3 after the reaction and inhibit the proliferation of Th0 cells blocked contact alloantigens after differentiation to Th1 direction.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392

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相關(guān)期刊論文 前1條

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