1，25-（OH）_2D_3及复方丹参对大鼠异基因骨髓移植后免疫耐受和造血重建的影响
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摘要
目的:本课题从基础研究入手,在建立大鼠异基因骨髓移植急性移植物抗宿主病(aGVHD)模型的基础上,用1,25-(OH)2D3及复方丹参作为干预方案,观察移植后大鼠aGVHD发生的严重程度、病理变化、造血恢复时间及T细胞亚群和细胞因子在体内的变化,探讨这种变化与相应事件之间的关系,并分析T淋巴细胞亚群和细胞因子对aGVHD的诊断价值及药物干预对其的影响。
方法:本研究分三部分;第一部分建立稳定的大鼠异基因骨髓移植急性移植物抗宿主病动物模型;第二部分1,25-(OH)2D3及复方丹参作为干预因素,对大鼠异基因骨髓移植后急性移植物抗宿主病和造血重建的影响;第三部分异基因骨髓移植后T细胞亚群和细胞因子的变化与免疫状态的关系。本研究采用随机对照研究、动态检测的方式来观察aGVHD组和干预组在大鼠异基因骨髓移植后aGVHD的程度、病理变化、造血恢复时间、体内细胞因子(IL-2和IFN-γ;IL-4和IL-10)以及T细胞亚群(CD4+、CD8+)的变化,用原位杂交的实验原理,在受鼠体内检测供鼠的Y染色体,以证实移植成功;用成熟的生物学方法(ELISA法)检测细胞因子,用免疫细胞学技术(流式细胞仪)检测T淋巴细胞亚群。
结果:第一部分结果显示:建立一个稳定的异基因骨髓移植急性移植物抗宿主病动物模型,需要加入脾细胞,骨髓细胞与脾细胞按1∶1及1∶1.5混合,大鼠aGVHD的发病时间相对集中于移植后的2～3周,中位生存时间分别为18.5天及16.5天,且全部出现较典型的aGVHD临床及病理表现。第二部分结果表明1,25-(OH)2D3组大鼠外周血白细胞、血红蛋白、血小板在不同时间点(第7,14,21天)的变化均高于aGVHD组,在移植后第21天白细胞、血红蛋白值均达到正常,血小板计数虽明显高于aGVHD组,差异有统计学意义(P<0.05),但仍未恢复正常;丹参组变化趋势与1,25-(OH)2D3组一致,且1,25-(OH)2D3组及复方丹参组组间比均无差异,联合组疗效并不优于单药组。本实验观察到尽管aGVHD组及干预组大鼠在50天观察期内全部死亡,但干预组与aGVHD组相比,其aGVHD的发病时间延迟,临床aGVHD评分降低,平均生存时间明显延长,差异有统计学意义(P<0.05),干预组大鼠的aGVHD的病理表现较aGVHD组轻。第三部分结果显示:aGVHD组CD4+值在移植后明显增加,与移植前相比差异有统计学意义(P<0.05),以第14天增高最为明显,第21天其数值接近移植前水平;CD8+的走势与CD4+相似, aGVHD组CD4+/CD8+的数值变化亦以第14天增高较为明显,T细胞亚群的变化与发生aGVHD的时间相一致;经干预处理后CD4+、CD8+增高的幅度较aGVHD组降低,差异有统计学意义(P<0.05),以CD4+更为明显,各干预组CD4+/CD8+的比值以第21天增高较为明显。
aGVHD组所测得的细胞因子IL-2、IFN-γ在移植后呈上升趋势,IL-10呈下降趋势,均以第14天为明显变化点,与第0天相比差异有统计学意义(P<0.05);第21天各指标接近移植前水平;IL-4在移植后各时间点其数值变化呈下降趋势;IL-2、IL-10、IFN-γ变化趋势与其在移植后第14天发生aGVHD相一致。
各干预组细胞因子IL-2、IFN-γ在移植后呈下降趋势,21天略有升高;IL-10则呈上升趋势,21天有所下降,与第0天相比均有统计学差异(P<0.05);IL-4各干预组在各时间点其数值变化呈上升趋势,变化幅度较小。各干预组经各自不同的干预方案预处理后,发生aGVHD的时间在21天左右,程度轻,IL-2、IL-10、IFN-γ的变化趋势与其一致。细胞因子在各时间点与aGVHD组比较差异有统计学意义。
结论:本研究成功建立全疆首个大鼠异基因骨髓移植aGVHD动物模型,骨髓细胞与脾细胞按1∶1及1∶1.5混合可以作为异基因骨髓移植后aGVHD研究的理想模型。将1,25-(OH)2D3及复方丹参作为干预因素,在大鼠异基因骨髓移植后能够促进造血重建并有预防aGVHD的作用;且干预方案能够明显降低CD4+T淋巴细胞的数量,调节Thl/Th2平衡,抑制Thl亚群(IL-2、IFN-γ)的增殖及功能发挥,促进Th2亚群(IL-10)的增殖,发挥免疫耐受的作用。细胞因子IL-4在本研究变化不显著,提示联合多个细胞因子和T细胞亚群的检测有助于免疫状态的判定。
Objective: The study began with fundamental research set up acute graft-versus-host disease(aGVHD) rat models of allogenic bone marrow transplantation, aimed to 1,25-(OH)2D3 and salvia miltiorrhiza as the interventional measures, to observe changes of aGVHD severity, physiology, hematopoietic recovery time, T cell subgroups and cytokines in rats after transplantation, to investigate the relationship between these changes and corresponding events and also to analyze the significance of T lymphocyte subgroups and cytokines to aGVHD diagnosis and influence of drug intervention.
Methods: The study consisted of three parts: firstly, establishing aGVHD animal models of allogenic bone marrow transplantation with rats; secondly, observing influence of 1,25-(OH)2D3 and salvia miltiorrhiza on aGVHD and hematopoietic reconstitution of rats after allogenic bone marrow transplantation; and thirdly, observing influence of drugs on immune state of bodies. Random control studies and dynamic detection were adopted to observe aGVHD severity, physiological changes, hematopoietic recovery time, changes of internal cytokines (IL-2 and IFN-γ; IL-4 and IL-10) and T cell subgroups (CD4+ and CD8+) of rats in the aGVHD group and the intervention groups after allogenic bone marrow transplantation; in-situ hybridization was used to detect Y chromosomes of donor rats in recipient rats to verify the success of transplantation; a mature biological method (ELISA) was employed to detect cytokines and immunocytology technology (flow cytometer) was used to detect T lymphocyte subgroups.
Results: Results of the first part indicated that spleen cells were required to establish a stable aGVHD animal mode of allogenic bone marrow transplantation. Bone marrow cells and spleen cells were mixed at 1∶1 and 1∶1.5, the attack time of aGVHD in rats was relatively centralized 2～3 weeks after transplantation; the median survival time was respectively 18.5 and 16.5 days and all subjects exhibited typical clinical and pathological manifestations of aGVHD. Results of the second part suggested that in the 1,25-(OH)2D3 group, changes of peripheral white blood cells, hemoglobin and platelets of rats were all higher than in the control group at different time points (the 7th, 14th and 21st day); on the 21st day after transplantation, results of white blood cell and hemoglobin examinations both reached normal, and the platelet count, though obviously higher than in the control group, didn’t return to the normal level. The change trend in the salvia group was consistent with that in the 1,25-(OH)2D3 group with no significant difference in comparison, and the therapeutic efficacy in the combined group was not better than in the single groups. It was found that all rats in the aGVHD group and intervention groups died in the 50-day study period, but the attack time of aGVHD was delayed in the intervention groups compared with that in the aGVHD group, the clinical aGVHD records of rats in the intervention groups were also lower than those of rats in the aGVHD groups, the average survival time was also markedly lengthened compared with that of rats in the aGVHD group with difference of statistical significance (P<0.05), and the pathological manifestation of aGVHD in rats of the intervention groups was milder than in the aGVHD group. Results of the third group showed that CD4+ value of the aGVHD group was significantly increased after transplantation with statistically significant difference in comparison with that before transplantation (P<0.05). The increase peaked on the 14th day and the value of the 21st day approximated to the level before transplantation; the trend of CD8+ was similar to that of CD4+; the CD4+/CD8+ value change of the aGVHD group was also the most notable on the 14th day and confirmed to the aGVHD onset time; after interventional treatment, the increase amplitudes of CD4+ and CD8+, CD4+ in particular, were lower than in the aGVHD group, and the CD4+/CD8+ value of each intervention group increased fairly remarkably on the 21st day.
In the aGVHD group, cytokines including IL-2 and IFN-γshowed increase trend while IL-10 showed decrease trend in examinations after transplantation. All of them displayed marked changes on the 14th day with statistically significant difference compared with the level before transplantation (P<0.05). All indexes approximated the levels before transplantation on the 21st day; after transplantation, the IL-4 value changes at each time point showed a decrease tendency; the change trends of IL-2, IL-10 and IFN-γwere in conformity with the fact that aGVHD occurred on the 14th day after transplantation.
Cytokines including IL-2 and IFN-γof each intervention group decreased after transplantation with a slight increase on the 21st day, but the IL-10 showed an increase tendency with a slight decrease on the 21st day. All of them had statistical difference in comparison with levels before the transplantation (P<0.05); in each intervention group, the IL-4 value change displayed an increase tendency at each time points, but the change amplitude was low. After pretreatment with different interventional measures, aGVHD occurred on around the 21st day with mild severity and the IL-2, IL-10 and IFN-γchanged in a synchronous tendency. Cytokines at each time point had statistically significant difference in compared with levels in the aGVHD group.
Conclusion: this study successfully setup the first aGVHD rat models of allogenic bone marrow transplantation in Xinjiang area. An ideal model can be created for studies on aGVHD after allogenic bone marrow transplantation when bone marrow cells and spleen cells are mixed at 1∶1 and 1∶1.5. 1,25-(OH)2D3 and salvia miltiorrhiza, as interventional factors, can promote hematopoietic reconstitution and prevent aGVHD in rats after allogenic bone marrow transplantation. The value of CD4+/CD8+ increases when aGVHD is severe and decreases when aGVHD is alleviated. Cytokines related to Th1, including IL-2 and IFN-γ, showed decrease tendencies generally in each intervention group, and the IL-10, a cytokine related to Th2, is in an increase tendency in general, indicating 1,25-(OH)2D3 and salvia miltiorrhiza have effects of regulating Th1/Th2 balance, inhibiting proliferation and function of Th1 subgroups, promoting proliferation of Th2 subgroups and acting for immune tolerance. No marked changes occurred to IL-4, suggesting detection of more than one cytokines and T cell subgroups is conducive to judgment of immune state.
方法:本研究分三部分;第一部分建立稳定的大鼠异基因骨髓移植急性移植物抗宿主病动物模型;第二部分1,25-(OH)2D3及复方丹参作为干预因素,对大鼠异基因骨髓移植后急性移植物抗宿主病和造血重建的影响;第三部分异基因骨髓移植后T细胞亚群和细胞因子的变化与免疫状态的关系。本研究采用随机对照研究、动态检测的方式来观察aGVHD组和干预组在大鼠异基因骨髓移植后aGVHD的程度、病理变化、造血恢复时间、体内细胞因子(IL-2和IFN-γ;IL-4和IL-10)以及T细胞亚群(CD4+、CD8+)的变化,用原位杂交的实验原理,在受鼠体内检测供鼠的Y染色体,以证实移植成功;用成熟的生物学方法(ELISA法)检测细胞因子,用免疫细胞学技术(流式细胞仪)检测T淋巴细胞亚群。
结果:第一部分结果显示:建立一个稳定的异基因骨髓移植急性移植物抗宿主病动物模型,需要加入脾细胞,骨髓细胞与脾细胞按1∶1及1∶1.5混合,大鼠aGVHD的发病时间相对集中于移植后的2～3周,中位生存时间分别为18.5天及16.5天,且全部出现较典型的aGVHD临床及病理表现。第二部分结果表明1,25-(OH)2D3组大鼠外周血白细胞、血红蛋白、血小板在不同时间点(第7,14,21天)的变化均高于aGVHD组,在移植后第21天白细胞、血红蛋白值均达到正常,血小板计数虽明显高于aGVHD组,差异有统计学意义(P<0.05),但仍未恢复正常;丹参组变化趋势与1,25-(OH)2D3组一致,且1,25-(OH)2D3组及复方丹参组组间比均无差异,联合组疗效并不优于单药组。本实验观察到尽管aGVHD组及干预组大鼠在50天观察期内全部死亡,但干预组与aGVHD组相比,其aGVHD的发病时间延迟,临床aGVHD评分降低,平均生存时间明显延长,差异有统计学意义(P<0.05),干预组大鼠的aGVHD的病理表现较aGVHD组轻。第三部分结果显示:aGVHD组CD4+值在移植后明显增加,与移植前相比差异有统计学意义(P<0.05),以第14天增高最为明显,第21天其数值接近移植前水平;CD8+的走势与CD4+相似, aGVHD组CD4+/CD8+的数值变化亦以第14天增高较为明显,T细胞亚群的变化与发生aGVHD的时间相一致;经干预处理后CD4+、CD8+增高的幅度较aGVHD组降低,差异有统计学意义(P<0.05),以CD4+更为明显,各干预组CD4+/CD8+的比值以第21天增高较为明显。
aGVHD组所测得的细胞因子IL-2、IFN-γ在移植后呈上升趋势,IL-10呈下降趋势,均以第14天为明显变化点,与第0天相比差异有统计学意义(P<0.05);第21天各指标接近移植前水平;IL-4在移植后各时间点其数值变化呈下降趋势;IL-2、IL-10、IFN-γ变化趋势与其在移植后第14天发生aGVHD相一致。
各干预组细胞因子IL-2、IFN-γ在移植后呈下降趋势,21天略有升高;IL-10则呈上升趋势,21天有所下降,与第0天相比均有统计学差异(P<0.05);IL-4各干预组在各时间点其数值变化呈上升趋势,变化幅度较小。各干预组经各自不同的干预方案预处理后,发生aGVHD的时间在21天左右,程度轻,IL-2、IL-10、IFN-γ的变化趋势与其一致。细胞因子在各时间点与aGVHD组比较差异有统计学意义。
结论:本研究成功建立全疆首个大鼠异基因骨髓移植aGVHD动物模型,骨髓细胞与脾细胞按1∶1及1∶1.5混合可以作为异基因骨髓移植后aGVHD研究的理想模型。将1,25-(OH)2D3及复方丹参作为干预因素,在大鼠异基因骨髓移植后能够促进造血重建并有预防aGVHD的作用;且干预方案能够明显降低CD4+T淋巴细胞的数量,调节Thl/Th2平衡,抑制Thl亚群(IL-2、IFN-γ)的增殖及功能发挥,促进Th2亚群(IL-10)的增殖,发挥免疫耐受的作用。细胞因子IL-4在本研究变化不显著,提示联合多个细胞因子和T细胞亚群的检测有助于免疫状态的判定。
Objective: The study began with fundamental research set up acute graft-versus-host disease(aGVHD) rat models of allogenic bone marrow transplantation, aimed to 1,25-(OH)2D3 and salvia miltiorrhiza as the interventional measures, to observe changes of aGVHD severity, physiology, hematopoietic recovery time, T cell subgroups and cytokines in rats after transplantation, to investigate the relationship between these changes and corresponding events and also to analyze the significance of T lymphocyte subgroups and cytokines to aGVHD diagnosis and influence of drug intervention.
Methods: The study consisted of three parts: firstly, establishing aGVHD animal models of allogenic bone marrow transplantation with rats; secondly, observing influence of 1,25-(OH)2D3 and salvia miltiorrhiza on aGVHD and hematopoietic reconstitution of rats after allogenic bone marrow transplantation; and thirdly, observing influence of drugs on immune state of bodies. Random control studies and dynamic detection were adopted to observe aGVHD severity, physiological changes, hematopoietic recovery time, changes of internal cytokines (IL-2 and IFN-γ; IL-4 and IL-10) and T cell subgroups (CD4+ and CD8+) of rats in the aGVHD group and the intervention groups after allogenic bone marrow transplantation; in-situ hybridization was used to detect Y chromosomes of donor rats in recipient rats to verify the success of transplantation; a mature biological method (ELISA) was employed to detect cytokines and immunocytology technology (flow cytometer) was used to detect T lymphocyte subgroups.
Results: Results of the first part indicated that spleen cells were required to establish a stable aGVHD animal mode of allogenic bone marrow transplantation. Bone marrow cells and spleen cells were mixed at 1∶1 and 1∶1.5, the attack time of aGVHD in rats was relatively centralized 2～3 weeks after transplantation; the median survival time was respectively 18.5 and 16.5 days and all subjects exhibited typical clinical and pathological manifestations of aGVHD. Results of the second part suggested that in the 1,25-(OH)2D3 group, changes of peripheral white blood cells, hemoglobin and platelets of rats were all higher than in the control group at different time points (the 7th, 14th and 21st day); on the 21st day after transplantation, results of white blood cell and hemoglobin examinations both reached normal, and the platelet count, though obviously higher than in the control group, didn’t return to the normal level. The change trend in the salvia group was consistent with that in the 1,25-(OH)2D3 group with no significant difference in comparison, and the therapeutic efficacy in the combined group was not better than in the single groups. It was found that all rats in the aGVHD group and intervention groups died in the 50-day study period, but the attack time of aGVHD was delayed in the intervention groups compared with that in the aGVHD group, the clinical aGVHD records of rats in the intervention groups were also lower than those of rats in the aGVHD groups, the average survival time was also markedly lengthened compared with that of rats in the aGVHD group with difference of statistical significance (P<0.05), and the pathological manifestation of aGVHD in rats of the intervention groups was milder than in the aGVHD group. Results of the third group showed that CD4+ value of the aGVHD group was significantly increased after transplantation with statistically significant difference in comparison with that before transplantation (P<0.05). The increase peaked on the 14th day and the value of the 21st day approximated to the level before transplantation; the trend of CD8+ was similar to that of CD4+; the CD4+/CD8+ value change of the aGVHD group was also the most notable on the 14th day and confirmed to the aGVHD onset time; after interventional treatment, the increase amplitudes of CD4+ and CD8+, CD4+ in particular, were lower than in the aGVHD group, and the CD4+/CD8+ value of each intervention group increased fairly remarkably on the 21st day.
In the aGVHD group, cytokines including IL-2 and IFN-γshowed increase trend while IL-10 showed decrease trend in examinations after transplantation. All of them displayed marked changes on the 14th day with statistically significant difference compared with the level before transplantation (P<0.05). All indexes approximated the levels before transplantation on the 21st day; after transplantation, the IL-4 value changes at each time point showed a decrease tendency; the change trends of IL-2, IL-10 and IFN-γwere in conformity with the fact that aGVHD occurred on the 14th day after transplantation.
Cytokines including IL-2 and IFN-γof each intervention group decreased after transplantation with a slight increase on the 21st day, but the IL-10 showed an increase tendency with a slight decrease on the 21st day. All of them had statistical difference in comparison with levels before the transplantation (P<0.05); in each intervention group, the IL-4 value change displayed an increase tendency at each time points, but the change amplitude was low. After pretreatment with different interventional measures, aGVHD occurred on around the 21st day with mild severity and the IL-2, IL-10 and IFN-γchanged in a synchronous tendency. Cytokines at each time point had statistically significant difference in compared with levels in the aGVHD group.
Conclusion: this study successfully setup the first aGVHD rat models of allogenic bone marrow transplantation in Xinjiang area. An ideal model can be created for studies on aGVHD after allogenic bone marrow transplantation when bone marrow cells and spleen cells are mixed at 1∶1 and 1∶1.5. 1,25-(OH)2D3 and salvia miltiorrhiza, as interventional factors, can promote hematopoietic reconstitution and prevent aGVHD in rats after allogenic bone marrow transplantation. The value of CD4+/CD8+ increases when aGVHD is severe and decreases when aGVHD is alleviated. Cytokines related to Th1, including IL-2 and IFN-γ, showed decrease tendencies generally in each intervention group, and the IL-10, a cytokine related to Th2, is in an increase tendency in general, indicating 1,25-(OH)2D3 and salvia miltiorrhiza have effects of regulating Th1/Th2 balance, inhibiting proliferation and function of Th1 subgroups, promoting proliferation of Th2 subgroups and acting for immune tolerance. No marked changes occurred to IL-4, suggesting detection of more than one cytokines and T cell subgroups is conducive to judgment of immune state.
引文
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