硼替佐米干预对Allo-HSCT后aGVHD进程的影响及机制研究

发布时间：2018-06-21 23:24

本文选题：移植物抗宿主病模型 + 异基因造血干细胞移植　；参考：《苏州大学》2011年博士论文

【摘要】：一、两种不同预处理方式建立小鼠异基因造血干细胞移植急性移植物抗宿主病(aGVHD)模型目的:利用两种不同的预处理方式建立小鼠异基因造血细胞移植aGVHD模型。方法:25只SPF级BALB/c小鼠分为5组,分别接受7、7.5、8、8.5、9Gy ~(60)Coγ射线全身照射。C57BL/6(H-2~b,♂)小鼠为供鼠,BALB/c小鼠(H-2~d,♀)为受鼠。20只BALB/c小鼠经预处理后随机分为4组,分别尾静脉输注1×10~6、2.5×10~6、5×10~6、10×10~6个骨髓细胞重建造血。在能重建造血的基础上建立aGVHD模型,输注10×10~6个骨髓细胞基础上再输注1×10~6、2.5×10~6、5×10~6或10×10~6个脾细胞。减弱强度的预处理方式用药物加小剂量的辐照方式,于移植前第8天开始腹腔注射氟达拉滨(fludarabine; 200mg/kg)至第4天,接着腹腔注射环磷酰胺(cyclophoshpamide; 60mg/kg)至移植前1天,最后在移植前~(60)Coγ射线全身照射(4Gy),建立aGVHD模型,观察小鼠生存状态及生存率。HE染色检测靶器官病理组织切片,流式细胞术检测小鼠嵌合度。结果:接受TBI 7,7.5,8,8.5和9Gy预处理的小鼠中,照射7Gy小鼠全部存活,照射7.5Gy小鼠中位生存期为31天,40天60%死亡。照射8Gy小鼠中位生存期为21天,40天内80%死亡。照射8.5Gy小鼠中位生存期为14天,40天内100%死亡。照射9Gy小鼠中位生存期为8天,40天内100%死亡。采用Log-rank检验分析生存时间,χ~2=24.72, P0.0001。输注5×10~6骨髓细胞可全部重建造血,100天生存率为100%。单纯输注骨髓细胞不会诱发aGVHD。低剂量脾细胞输注小鼠aGVHD程度轻,40%出现aGVHD性相关死亡;5×10~6剂量脾细胞输注小鼠100%出现aGVHD相关死亡,疾病严重程度属于中等,中位生存期为19天。10×10~6剂量脾细胞输注小鼠100%出现aGVHD相关死亡,疾病严重程度属于重度。5×10~6剂量脾细胞组出现典型的aGVHD病理表现,21天后为完全供体嵌合状态。减弱强度的预处理方式建立的模型,选用中等强度的脾细胞(5×10~6),骨髓细胞为(1×10~7);其中位生存期为18天,21天后为供受体混合嵌合状态。结论:8.5Gy TBI对于BALB/c小鼠是清髓性照射剂量。预处理后输注5×10~6个骨髓细胞可全部重建造血。输注5×10~6个脾细胞可诱发中度程度aGVHD;而输注10×10~6个脾细胞可诱发严重程度aGVHD。二、硼替佐米对宿主DC的调节作用及其对aGVHD的影响目的:蛋白酶体抑制剂硼替佐米移植后早期注射(移植后0-2天)可以预防aGVHD的发生。本研究旨在探讨硼替佐米对宿主DC的调节作用及其对aGVHD的影响。揭示硼替佐米在移植后早期注射延缓aGVHD是通过其对DC成熟与功能的抑制作用实现的。方法:体外在GM-CSF和IL-4条件下利用骨髓来源培养的DC,经过不同浓度硼替佐米处理后,流式细胞术检测协同共刺激分子CD40、CD80、CD86及MHC-II类抗原表达。用经过不同浓度硼替佐米处理后的DC加入OVA肽段,与OT-I小鼠磁珠分离CD8+T细胞共育。胞内染色观察CD8+T细胞IL-2、IFN-γ和TNF-α表达情况,同时检测上清中上述三种细胞因子的分泌量。EMSA检测经过不同浓度硼替佐米预处理的DC的NF-κB的入核活性。利用骨髓来源的培养DC经过不同浓度硼替佐米预处理后的DC与异基因小鼠脾细胞共育,[3H]-TdR掺入法检测硼替佐米处理后DC对异基因淋巴细胞增殖的影响。体内利用硼替佐米预处理的宿主DC输入到aGVHD小鼠体内,观察小鼠生存状态及生存率。结果:小鼠骨髓来源的细胞在GM-CSF(10ng/mL)和IL-4(1 ng/mL)培养7天后CD11c阳性率为85%以上。经过不同浓度硼替佐米处理的DC在LPS刺激后,CD40、CD80、CD86及MHC-II类抗原表达下调,呈现出明显的剂量依赖性。经过硼替佐米处理的DC与OT-I CD8+T细胞共育后检测胞内细胞因子流式图显示:CD8+TNF-α+T和CD8+INF-γ+T细胞数目逐渐减少,呈现出剂量依赖性。细胞上清细胞因子检测显示:CD8+T分泌TNF-α和INF-γ量逐渐减少,而IL-2的水平并无明显变化。EMSA结果显示,经过硼替佐米预处理的imDC在LPS刺激条件下NF-κB入核量减少,并且呈现出浓度依赖性。经过不同浓度硼替佐米处理的DC与异基因脾细胞进行MLR实验结果显示:DC在经硼替佐米(2nM、10nM和50nM)处理后,其激活异基因T细胞增殖的能力随着硼替佐米浓度的增高而逐渐减弱。经过50nM硼替佐米处理后的1×10~6宿主DC输入aGVHD模型小鼠后,其生存率要明显高于未用硼替佐米处理而过继输入DC的aGVHD小鼠模型。结论:经过硼替佐米处理的DC其成熟受到抑制,继而其抗原递呈能力下调,导致激活同种异基因淋巴细胞的增殖能力减弱。主要机制是抑制了NF-κB的入核活性。移植后早期注射硼替佐米可能使宿主DC功能下调,降低了异基因抗原的递呈,从而预防了aGVHD的发生。三、TLR4通路与IL-1β在硼替佐米延迟注射促进aGVHD过程中的作用目的:蛋白酶体抑制剂硼替佐米移植后延迟注射(移植后3天以后)可以促进aGVHD的发生。本研究旨在探讨TLR4通路与IL-1β在延迟注射硼替佐米促进GVHD中的作用。方法:体外系统模拟延迟注射与即时注射硼替佐米对DC和巨噬细胞细胞因子分泌的影响。培养骨髓来源的C57BL/6背景DC,于培养第7天LPS刺激,在LPS刺激前或刺激后加入硼替佐米。实验分四组:①延迟硼替佐米组:LPS(100ng/mL)刺激6 h后,加入不同浓度硼替佐米共育,24 h后检测细胞因子浓度。②即时硼替佐米组:先加入不同浓度硼替佐米,6 h后再加入LPS (100ng/mL)刺激。③LPS对照组:直接加LPS刺激。④硼替佐米对照组:直接加不同浓度硼替佐米共育。利用上述方法处理的DC与异基因淋巴细胞进行MLR反应,[3H]-TdR掺入法检测硼替佐米处理后DC对T淋巴细胞增殖影响; ELISA法同时检测上清中细胞因子TNF-α和IL-1β水平。体内采取三种不同的方式对aGVHD动物模型进行干预,研究TLR4通路与硼替佐米延迟干预互作对aGVHD进程的影响。 (1)阻断TLR4通路对硼替佐米延迟注射促进aGVHD的影响。用TLR4 KO小鼠做为受体,BALB/c为供体,单纯辐照法(9.0Gy)建立小鼠GVHD模型,实验分即时注射硼替佐米组、延迟注射硼替佐米TLR4 KO组、延迟注射硼替佐米C57BL/6组、aGVHD C57BL/6模型对照组和aGVHD TLR4 KO模型对照组,观察各组小鼠注射硼替佐米后的生存状态及生存率。 (2)移植前预处理产生的不同LPS水平对硼替佐米延迟注射促进aGVHD的影响。运用氟达拉滨加小剂量照射(4Gy)预处理方式建立小鼠aGVHD模型,检测不同时间小鼠体内的LPS水平;观察小鼠延迟注射硼替佐米生存率;同时根据体外模拟实验结果设上调体内LPS浓度实验,分GVHD模型对照组,延迟注射硼替佐米组,LPS+延迟注射硼替佐米组(建模第3天给GVHD模型鼠先腹腔注射LPS(5 mg/kg),6 h后注射硼替佐米1次)和LPS对照组(实验第3天给GVHD模型鼠注射LPS(5 mg/kg) 1次)。观察小鼠生存状态及生存率。 (3)阻断炎性因子IL-1β对硼替佐米延迟注射促进aGVHD的影响。大剂量辐照法建立小鼠aGVHD模型:选用SPF级接受8.5Gy致死剂量~(60)Co辐照的BALB/c小鼠作为受鼠,尾静脉移植入C57BL/6小鼠1.0×10~7骨髓细胞和0.5×10~7脾细胞,建立小鼠aGVHD模型。骨髓移植后第1,3和5天取血,LAL法检测小鼠LPS血清水平;同时检测延迟注射硼替佐米组(骨髓移植后第3天注射,连续2天)、即时注射硼替佐米组(骨髓移植后连续注射2天)和GVHD模型组的Th1 (IFN-γ),Th2 (IL-4)、TNF-α与IL-1β血清水平。用上述方法建立小鼠aGVHD模型,实验分即时注射硼替佐米组、延迟注射硼替佐米组、阿那白滞素阻断IL-1β组(骨髓移植后连续注射2天阿那白滞素)和阿那白滞素阻断IL-1β+延迟注射硼替佐米组(骨髓移植后注射阿那白滞素阻滞IL-1β2天,第3天注射硼替佐米)。于延迟注射硼替佐米后小鼠濒死时免疫组织化学法检测各组小肠TNFR的表达;ELISA法检测血清细胞因子浓度;计算GVHD积分;观察GVHD各组各个脏器(肝脏、皮肤、肺及小肠)HE染色病理组织切片及生存率。结果:原代培养的DC在先LPS刺激6 h后与不同浓度硼替佐米的作用下比单用LPS刺激分泌的IL-1β显著增高(P0.01);而TNF-α则无上述现象。我们用同样的方法也检测了巨噬细胞,未发现差异。经延迟和即时处理的DC与同种异基因小鼠的脾细胞共育进行MLR实验,[3H]-TdR法检测异结果显示:较高浓度硼替佐米(50nM和10nM)延迟处理的DC其激活同种异基因淋巴细胞的能力要比LPS组和即时组都要强(P0.01)。延迟模拟组IL-4水平有显著意义上升(P0.05)。延迟给药aGVHD小鼠血清细胞因子浓度结果显示:延迟注射硼替佐米aGVHD组与aGVHD模型组或即时注射硼替佐米aGVHD组比,移植后第4天小鼠IL-1β和IFN-γ水平升高。 TLR4敲基因建立的aGVHD模型,在延迟注射硼替佐米后不会立即死亡,且其生存率要显著高于C57BL/6对照组和TLR4 KO模型对照组;而C57BL/6延迟注射组小鼠会立即死亡。运用氟达拉滨加小剂量辐照法与大剂量单独辐照法建立的小鼠aGVHD模型相比,前者早期体内LPS升高不明显。不同预处理产生的LPS水平不同,在氟达拉滨加小剂量辐照组骨髓移植后,第1,3和5天的LPS水平分别为(0.17±0.02 )Eu/mL、(0.21±0.04 )Eu/mL和(0.23±0.05)Eu/mL;而大剂量辐照预处理组则为(0.27±0.02)Eu/mL、(0.47±0.02)Eu/mL和(0.69±0.04)Eu/mL。两种方式比较结果:大剂量辐照组LPS浓度显著高于氟达拉滨加小剂量辐照组(P0.01)。氟达拉滨加小剂量辐照组LPS水平相对稳定,而大剂量辐照组则呈现出逐渐上升趋势。运用氟达拉滨与小剂量照射预处理方式建立的小鼠GVHD模型在延迟注射硼替佐米后生存期明显比大剂量辐照组延长;在上调LPS实验中,LPS+延迟注射硼替佐米组与LPS对照组存在明显差异,肠道肿胀,炎症表现。辐照法建立aGVHD模型后,用阿那白滞素阻断IL-1β可以明显延长延迟注射硼替佐米小鼠的生存率。血清中IFN-γ与TNF-α下降,IL-4上升。组织病理变化、体重变化、体内炎性因子变化均有差异。结论:移植后期预处理造成肠道损伤,LPS激活TRL4通路,此时注射硼替佐米促进了IL-1β的分泌,放大“炎性风暴”,继而过度激活宿主DC细胞及供者T细胞而促进GVHD进程。证明了TLR4信号通路激活是引发硼替佐米延迟注射导致GVHD相关性死亡的重要原因;并研究不同的预处理方式会导致机体产生不同水平的LPS,从而激活TLR4通路信号的强度不同,最终在延迟注射硼替佐米促进GVHD相关性死亡中起到重要作用;IL-1β在延迟注射硼替佐米促进aGVHD的进程中起到了放大“炎性风暴”的关键作用。
[Abstract]:One, two different preconditioning methods to establish acute graft-versus-host disease (aGVHD) model in mice with allogeneic hematopoietic stem cell transplantation
Objective: to establish a mouse model of allogeneic hematopoietic cell transplantation (aGVHD) using two different pretreatment methods.
Methods: 25 SPF grade BALB/c mice were divided into 5 groups. The mice were treated with 7,7.5,8,8.5,9Gy ~ (60) Co gamma ray whole body irradiation.C57BL/6 (H-2~b) mice. The BALB/c mice were randomly divided into 4 groups after pretreatment, and the tail vein infusion 1 x 10~6,2.5 * 10~6,5 * * * * * * bone marrow cells rebuilt hematopoiesis. On the basis of the reconstruction of hematopoiesis, the aGVHD model was established. On the basis of infusion of 10 x 10~6 bone marrow cells, 1 x 10~6,2.5 x 10~6,5 x 10~6 or 10 x 10~6 spleen cells were reinjected. The preconditioning methods of weakened intensity were treated with drugs plus small dose of irradiation, and the intraperitoneal injection of fluda La La (fludarabine; 200mg/kg) to fourth days before the transplantation, and then to fourth days before transplantation. By intraperitoneal injection of cyclophosphamide (cyclophoshpamide; 60mg/kg) to 1 days before transplantation, the aGVHD model was established before ~ (60) Co gamma ray whole body irradiation (4Gy) before transplantation. The survival state and survival rate of mice were observed by.HE staining to detect the pathological tissue section of the target organ, and the chimerism of mice was detected by flow cytometry.
Results: in the mice pretreated with TBI 7,7.5,8,8.5 and 9Gy, all the mice irradiated with 7Gy were alive, the median survival time of the irradiated 7.5Gy mice was 31 days, and the 40 day 60% died. The median survival time of the irradiated 8Gy mice was 21 days and 80% died within 40 days. The median survival time of the irradiated 8.5Gy mice was 14 days and 100% died in 40 days. The median survival period of the irradiated 9Gy mice was 8 days, 40. 100% days of death in the day. Using Log-rank test to analyze survival time, X ~2=24.72, P0.0001. infusion of 5 x 10~6 bone marrow cells can all rebuild hematopoiesis, 100 natural survival rate is 100%. simply infusion of bone marrow cells will not induce aGVHD. low dose spleen cell infusion mice aGVHD degree, 40% aGVHD related death, 5 x 10~6 dose of spleen cells transfused 10 mice. 0% the incidence of aGVHD related death, the severity of the disease was moderate, the median survival time was 19 days and the dose of.10 x 10~6 in the spleen cell infusion mice 100% appeared aGVHD related death, the severity of the disease was a severe.5 * 10~6 dose of spleen cell group, the typical aGVHD pathological manifestation, 21 days after the complete donor chimerism.
A moderate intensity of splenocytes (5 x 10~6), and bone marrow cells (1 x 10~7), were established with a moderate intensity preconditioning model. The survival period was 18 days and the donor and receptor mixed chimerism was 21 days later.
Conclusion: 8.5Gy TBI is a myeloamedullary dose for BALB/c mice. After preconditioning, 5 x 10~6 bone marrow cells can reconstruct all hematopoiesis. The infusion of 5 x 10~6 splenocytes can induce moderate degree of aGVHD, while 10 x 10~6 transfused splenocytes can induce severity aGVHD..
Two, the regulatory effect of bortezomib on host DC and its effect on aGVHD.
Objective: the early injection of the proteasome inhibitor bortel Zomi (0-2 days after transplantation) could prevent the occurrence of aGVHD. The aim of this study was to investigate the effect of bortel Zomi on the host DC and its effect on aGVHD. It was revealed that the early injection of bortel Zomi delayed the inhibition of the maturation and function of DC by the delayed injection of aGVHD.
Methods: in vitro, DC was cultured with bone marrow from GM-CSF and IL-4. After different concentrations of bortezomib, flow cytometry was used to detect co stimulator CD40, CD80, CD86 and MHC-II antigen expression. OVA peptide was added to DC after different concentrations of bortezomib, and CD8+T cells were separated from OT-I mouse magnetic beads. The expression of IL-2, IFN- gamma and TNF- alpha in CD8+T cells was observed by internal staining, and the secretion of the above three cytokines in the supernatant was detected by.EMSA to detect the nucleation activity of DC NF- kappa B pretreated with bortezomib at different concentrations. DC and allogeneic mouse splenocytes pretreated with different concentrations of bortezomib were used to culture DC from bone marrow. The effect of DC on the proliferation of allogeneic lymphocytes after bortezomizo treatment was detected by [3H]-TdR incorporation, and the host DC pretreated with bortezomizomi was injected into the body of aGVHD mice to observe the survival and survival rate of mice.
Results: the positive rate of CD11c in mice bone marrow derived cells was more than 85% after 7 days of GM-CSF (10ng/mL) and IL-4 (1 ng/mL) culture. After DC stimulated by different concentrations of bortezomib in LPS, the expression of CD40, CD80, CD86 and MHC-II antigens down was down. The post cell cytokine flow pattern showed that the number of CD8+TNF- alpha +T and CD8+INF- gamma +T cells decreased gradually and showed a dose-dependent manner. Cell supernatant cytokine detection showed that CD8+T secretion of TNF- A and INF- gamma decreased gradually, but the level of IL-2 had no obvious change in.EMSA results, and imDC of bortezomizomi pretreated in LPS stimulation. The nucleation of NF- kappa B decreased and showed a concentration dependence. The results of MLR experiment with DC and allogeneic splenocytes treated with bortezomib at different concentrations showed that the ability of DC to activate the proliferation of the allogeneic T cells gradually weakened with the increase of bortezomizomi (2nM, 10nM and 50nM). After 50nM, the proliferation of the allogeneic cells gradually weakened. After bortezomizomi treated 1 x 10~6 host DC input aGVHD model mice, the survival rate was significantly higher than that of the aGVHD mouse model that was adoptive to input DC without bortezomizomizomi treatment.
Conclusion: the maturity of DC treated by bortezomizomi was inhibited, and the ability of antigen presentation was down, leading to the reduction of the proliferation ability of the allogeneic lymphocyte activation. The main mechanism was to inhibit the nucleation activity of NF- kappa B. And it prevented the occurrence of aGVHD.
Three, the role of TLR4 pathway and IL-1 beta in the delayed injection of bortezomib in promoting aGVHD.
Objective: the delayed injection of bortezomib after transplantation (3 days after transplantation) can promote the occurrence of aGVHD. The purpose of this study was to explore the role of TLR4 pathway and IL-1 beta in the delayed injection of bortezomib to promote GVHD.
Methods: the effects of delayed injection and immediate injection of bortezomib on the cytokine secretion of DC and macrophages were simulated in vitro. The C57BL/6 background DC of bone marrow was cultured for seventh days of LPS stimulation, and bortezomib was added before or after LPS stimulation. The experiment was divided into four groups: delayed bortezomib group: LPS (100ng/mL) stimulated 6 h and added no With the same concentration borosilia for Zomi, the concentration of cytokine was detected after 24 h. (2) immediate borosilia in the Zomi group: first adding different concentrations of borteto Zomi, and then adding LPS (100ng/mL) stimulation after 6 h. (3) LPS control group: direct plus LPS stimulation. (4) bortel control group: direct plus different concentrations of boron for Zomi co breeding. Using the above method, DC and allogenein treatment treated DC and allogeneic drenching were used. The effect of DC on the proliferation of T lymphocyte after bortezomizomi treatment was detected by MLR reaction, and the level of TNF- alpha and IL-1 beta in the supernatant was detected by ELISA.
In vivo, three different ways were used to intervene the aGVHD animal model, and the effect of delayed interaction between TLR4 pathway and bortezomib on the aGVHD process was studied.
(1) blocking the effect of TLR4 pathway on the delayed injection of bortezomib. Using TLR4 KO mice as the receptor, BALB/c as the donor and the simple irradiation (9.0Gy), the mouse GVHD model was established. The experiment was divided into the bortezomizomi group, the delayed injection of bortezomizomi TLR4 KO group, the delayed injection of bortezomizomi C57BL/6 group, the aGVHD C57BL/6 model control group and the control group. The survival status and survival rate of mice in each group were observed after injection of bortezomib by D TLR4 KO model control group.
(2) the effect of different LPS levels on the delayed injection of bortezomib on the effect of delayed injection of bortezomib on aGVHD. A mouse aGVHD model was established by preconditioning with fluatoman and small dose of irradiation (4Gy) to detect the LPS level in mice at different time, and to observe the survival rate of bortezomi in the delayed injection of mice; meanwhile, the experimental results were based on the experimental results in vitro. We set up the LPS concentration test in the body, divided the GVHD model control group, delayed injection of bortezomib group, LPS+ delayed injection of bortezomib group (LPS (5 mg/kg) first intraperitoneal injection of GVHD model rats for third days, 6 h after injection of bortezomib) and LPS control group (the experiment third days to GVHD model mice injected LPS (5 mg/kg) 1 times). Survival rate.
(3) blocking the effect of inflammatory factor IL-1 beta on the delayed injection of bortezomib to promote aGVHD. The mice aGVHD model was established by large dose irradiation. The BALB/c mice irradiated with 8.5Gy lethal dose of ~ (60) Co irradiation were selected as the mice, the tail vein was transplanted into the C57BL/6 mice of 1 x 10~7 marrow cells and 0.5 x 10~7 splenocytes, and the mouse aGVHD model was established. Blood was taken at 1,3 and 5 days after transplantation, and the serum level of LPS in mice was detected by LAL, and the delayed injection of bortezomizomi group (third days after bone marrow transplantation for 2 days), immediate injection of bortezomizomi group (2 days after bone marrow transplantation) and Th1 (IFN- gamma) of GVHD model group, Th2 (IL-4), TNF- alpha and IL-1 beta serum level. The rat aGVHD model was divided into the bortezomizomi group, delayed injection of bortezomib group, ananbin blocking the IL-1 beta group (2 days of ananlis after bone marrow transplantation) and ananwhite blocking the IL-1 beta + delayed injection of bortezomizomi group (IL-1 beta blockage after bone marrow transplantation was injected into IL-1 beta after the bone marrow transplantation, and bortezomib was injected third days). The expression of TNFR in small intestine was detected by immuno histochemical method when Yu Yanchi was injected with bortezomib, and the serum cytokine concentration was detected by ELISA, and GVHD integral was calculated. The pathological tissue section and survival rate of HE staining in various organs of GVHD (liver, skin, lung and small intestine) were observed.
Results: the primary culture of DC before LPS stimulated 6 h with different concentrations of bortezomib, which was significantly higher than the IL-1 beta secreted by single LPS (P0.01), while TNF- alpha had no above phenomenon. We also detected the macrophages with the same method. The delayed and immediately treated DC and allogeneic mice had splenocytes altogether. MLR test and [3H]-TdR assay showed that the ability of DC to activate allogeneic lymphocytes at high concentration of bortezomizomi (50nM and 10nM) was stronger than that of LPS and immediate groups (P0.01). There was a significant increase in IL-4 level in the delayed simulation group (P0.05).
The serum cytokine concentration of delayed aGVHD mice showed that the level of IL-1 beta and IFN- gamma in the delayed injection of bortezomizomi group aGVHD was compared with the aGVHD model group or the immediate injection of bortezomizomi aGVHD group. The level of IL-1 beta and IFN- gamma in mice was increased fourth days after the transplantation.
The aGVHD model, established by TLR4 knockout, did not die immediately after delayed injection of bortezomib, and its survival rate was significantly higher than that of the C57BL/6 control group and the TLR4 KO model control group, while the C57BL/6 delayed injection group died immediately.
Compared with the aGVHD model established by the small dose of fludarabine plus small dose irradiation and the large dose of single irradiation, the early body LPS in the former was not significantly elevated. The levels of LPS produced by different pretreatments were different, and the LPS levels of 1,3 and 5 days were (0.17 + 0.02) Eu/mL and (0.21 + 0.04) Eu/mL respectively after bone marrow transplantation in fludarabine plus small dose irradiation group. And (0.23 + 0.05) Eu/mL, while large dose radiation pretreatment group was (0.27 + 0.02) Eu/mL, (0.47 + 0.02) Eu/mL and (0.69 + 0.04) Eu/mL. two. The concentration of LPS in large dose irradiation group was significantly higher than that of fludararin plus small dose irradiation group (P0.01). The level of LPS in fludarbin plus small dose irradiation group was relatively stable, while the large dose irradiation group was relatively stable. The GVHD model of mice established by preconditioning with fludarabine and small dose of bortezomib was significantly longer than the large dose irradiation group after the delayed injection of bortezomib. In the up regulation of LPS, the LPS+ delayed injection of bortezomizomi group was significantly different from that of the LPS control group, the intestinal swelling and inflammation.
After the aGVHD model was established by irradiation, the survival rate of delayed injection of bortezomib was significantly prolonged by blocking the IL-1 beta with an Alban blockage. Serum IFN- and TNF- alpha were significantly increased.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392

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