IL-2上调淋巴细胞SOCS-3表达阻滞Th1分化抑制小鼠急性GVHD的实验研究

发布时间：2018-01-11 07:19

本文关键词：IL-2上调淋巴细胞SOCS-3表达阻滞Th1分化抑制小鼠急性GVHD的实验研究　出处：《复旦大学》2007年博士论文　论文类型：学位论文

更多相关文章： IL-2 SOCS-3 急性GVHD Th1细胞

【摘要】： 目的:研究小剂量IL-2预孵育移植物后对小鼠急性GVHD的影响,探讨小剂量IL-2预孵育后同种异基因淋巴细胞中SOCS-3蛋白表达的变化在此过程中的作用与机制。方法: 1、用IL-2预孵育DO11.10转基因小鼠naive T淋巴细胞、C57BL/6N小鼠脾细胞和C57BL/6N小鼠na(?)veCD4+T细胞,检测SOCS-3基因的表达变化。用OVA_(323-329)特异性抗原刺激活化DO11.10TCR转基因小鼠T细胞,检测T淋巴细胞经抗原活化后SOCS-3基因的表达变化。 2、构建pLX-SOCS3-SN载体,建立高表达SOCS-3基因的DO11.10细胞(DS细胞),用OVA_(323-329)特异性抗原刺激活化DS细胞,检测DS细胞的免疫应答能力。 3、用IL-2(50U/ml)预孵育DO11.10 TCR转基因小鼠na(?)ve T淋巴细胞、C57BL/6N小鼠脾细胞和C57BL/6N小鼠na(?)ve CD4+T细胞,然后用OVA_(323-329)特异性抗原和BALB/C小鼠异基因抗原分别对其进行刺激,测定DO11.10 TCR转基因小鼠na(?)ve T淋巴细胞、C57BL/6N小鼠脾细胞和C57BL/6N小鼠na(?)ve CD4+T淋巴细胞免疫增殖能力。 4、以C57BL/6N小鼠的T淋巴细胞为反应细胞,以BALB/C小鼠灭活的脾细胞为刺激细胞。将反应细胞在终浓度为50U/ml的IL-2中预孵育4小时,然后与刺激细胞在细胞因子IL-12或者IL-4存在的情况下进行混合淋巴细胞反应。与对照组(即反应细胞不给予IL-2预孵育4小时)比较第14天时IL-12R_(β1)、IL-12R_(β2)和胞内IFN-γ和IL-4的表达的差异,进行统计学检验。 5、受鼠为雌性BALB/C,预处理为~(137)Se TBI 5GY,分为A、B、C、D、E共5组(n=12),分别过继性植入雄性C57BL/6N脾淋巴细胞5×10~7、1××10~7和雄性BALB/C×C57BL/6N→F1脾淋巴细胞0.5×10~7、1×10~7、5×10~7(个/只)。对存活时间超过21天的受鼠在不同时间点用PCR检测供者淋巴细胞嵌合体,观察受鼠生存时间和急性GVHD的症状。 6、受鼠为雌性BALB/C,预处理为~(137)Se TBI 5GY,供鼠为雄性C57BL/6N。每只受鼠均腹腔注射供鼠脾淋巴细胞3×10~7个,受鼠分为A(直接植入供鼠淋巴细胞)、B(植入的淋巴细胞经过终浓度为50 U/ml的IL-2预孵育4小时)、C(植入的淋巴细胞先与灭活的BALB/C脾淋巴细胞反应72小时再植入)、D (植入的淋巴细胞先经过终浓度为50 U/ml的IL-2预孵育4小时,然后与灭活的BALB/C脾淋巴细胞混合淋巴细胞反应72小时再植入)共4组(n=9)。观察期为60天,观察受鼠生存时间和急性GVHD的症状,死亡受鼠均行肝脏和小肠的病理检查了解急性GVHD的病理改变情况。观察期结束时取每只小鼠脾细胞检测嵌合体,取肝脏和小肠行病理检查,了解急性GVHD的病理改变。结果: 1、DO11.10 TCR转基因小鼠na(?)ve T淋巴细胞、C57BL/6N小鼠脾细胞和C57BL/6小鼠na(?)ve CD4+T淋巴细胞经IL-2预孵育后,SOCS-3的表达明显升高,在4-6小时达到高峰。DO11.10 TCR转基因小鼠T细胞经OVA_(323-329)特异性抗原刺激后SOCS-3的表达明显降低,以2-3天最低。 2、高表达SOCS-3的DS细胞对OVA_(323-329)特异性抗原免疫应答能力明显减弱。 3、DO11.10转基因小鼠na(?)ve T淋巴细胞、C57BL/6N小鼠脾细胞和C57BL/6N小鼠na(?)ve CD4+T淋巴细胞经IL-2预孵育后对OVA_(323-329)特异性抗原和同种异基因抗原免疫增殖能力明显减弱。 4、经IL-2预孵育后的CD4+T淋巴细胞,接受异基因抗原刺激的同时加入外源性IL-12培养,第14天时,IL-12R_(β1)、IL-12R_(β2)的表达虽然呈阳性,但是明显低于未经预孵育组(P值0.05),IFN-γ与IL-4阳性率的比值也低于未经预孵育组(P0.05)。加入外源性IL-4培养时,经IL-2预孵育组IL-12R_(β1)、IL-12R_(β2)的表达也明显明低于未经预孵育组(P0.05),IL-4的表达明显要高于未经预孵育组(P0.05)。 5、TBI为5GY的免疫抑制后,在受鼠保留自身造血的情况下,过继性同种异基因淋巴细胞可以植活。MHC分子半相合的植活率(D组)比完全不相合的(B组)高,植活时间长(P0.01);在半相合的植入模型中植入的细胞量越多,植活率越高,植活时间逐渐延长(P0.05)。同时仍有急性GVHD发生,MHC完全不相合(A组)移植小鼠可发生致死性急性GVHD,半相合(E组)急性GVHD明显减轻(P0.01)。 6、IL-2(50 U/ml)预孵育同种异基因淋巴细胞4小时后,然后与灭活后的BALB/C脾淋巴细胞混合反应72小时,再过继性植入后其急性GVHD的发生强度明显减轻。结论: 小剂量的IL-2预孵育同种异基因的淋巴细胞后,可以明显减轻过继性植入后急性GVHD的发生。其机制与小剂量IL-2预孵育异基因淋巴细胞上调SOCS-3的表达后,抑制其增殖反应和阻滞Th0细胞接触同种抗原后向Th1方向的分化有关。
[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.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392

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