本文選題：移植物抗宿主病模型 + 異基因造血干細(xì)胞移植　； 參考：《蘇州大學(xué)》2011年博士論文

【摘要】：一、兩種不同預(yù)處理方式建立小鼠異基因造血干細(xì)胞移植急性移植物抗宿主病(aGVHD)模型 目的:利用兩種不同的預(yù)處理方式建立小鼠異基因造血細(xì)胞移植aGVHD模型。 方法:25只SPF級(jí)BALB/c小鼠分為5組,分別接受7、7.5、8、8.5、9Gy ~(60)Coγ射線全身照射。C57BL/6(H-2~b,♂)小鼠為供鼠,BALB/c小鼠(H-2~d,♀)為受鼠。20只BALB/c小鼠經(jīng)預(yù)處理后隨機(jī)分為4組,分別尾靜脈輸注1×10~6、2.5×10~6、5×10~6、10×10~6個(gè)骨髓細(xì)胞重建造血。在能重建造血的基礎(chǔ)上建立aGVHD模型,輸注10×10~6個(gè)骨髓細(xì)胞基礎(chǔ)上再輸注1×10~6、2.5×10~6、5×10~6或10×10~6個(gè)脾細(xì)胞。減弱強(qiáng)度的預(yù)處理方式用藥物加小劑量的輻照方式,于移植前第8天開(kāi)始腹腔注射氟達(dá)拉濱(fludarabine; 200mg/kg)至第4天,接著腹腔注射環(huán)磷酰胺(cyclophoshpamide; 60mg/kg)至移植前1天,最后在移植前~(60)Coγ射線全身照射(4Gy),建立aGVHD模型,觀察小鼠生存狀態(tài)及生存率。HE染色檢測(cè)靶器官病理組織切片,流式細(xì)胞術(shù)檢測(cè)小鼠嵌合度。 結(jié)果:接受TBI 7,7.5,8,8.5和9Gy預(yù)處理的小鼠中,照射7Gy小鼠全部存活,照射7.5Gy小鼠中位生存期為31天,40天60%死亡。照射8Gy小鼠中位生存期為21天,40天內(nèi)80%死亡。照射8.5Gy小鼠中位生存期為14天,40天內(nèi)100%死亡。照射9Gy小鼠中位生存期為8天,40天內(nèi)100%死亡。采用Log-rank檢驗(yàn)分析生存時(shí)間,χ~2=24.72, P0.0001。輸注5×10~6骨髓細(xì)胞可全部重建造血,100天生存率為100%。單純輸注骨髓細(xì)胞不會(huì)誘發(fā)aGVHD。低劑量脾細(xì)胞輸注小鼠aGVHD程度輕,40%出現(xiàn)aGVHD性相關(guān)死亡;5×10~6劑量脾細(xì)胞輸注小鼠100%出現(xiàn)aGVHD相關(guān)死亡,疾病嚴(yán)重程度屬于中等,中位生存期為19天。10×10~6劑量脾細(xì)胞輸注小鼠100%出現(xiàn)aGVHD相關(guān)死亡,疾病嚴(yán)重程度屬于重度。5×10~6劑量脾細(xì)胞組出現(xiàn)典型的aGVHD病理表現(xiàn),21天后為完全供體嵌合狀態(tài)。 減弱強(qiáng)度的預(yù)處理方式建立的模型,選用中等強(qiáng)度的脾細(xì)胞(5×10~6),骨髓細(xì)胞為(1×10~7);其中位生存期為18天,21天后為供受體混合嵌合狀態(tài)。 結(jié)論:8.5Gy TBI對(duì)于BALB/c小鼠是清髓性照射劑量。預(yù)處理后輸注5×10~6個(gè)骨髓細(xì)胞可全部重建造血。輸注5×10~6個(gè)脾細(xì)胞可誘發(fā)中度程度aGVHD;而輸注10×10~6個(gè)脾細(xì)胞可誘發(fā)嚴(yán)重程度aGVHD。 二、硼替佐米對(duì)宿主DC的調(diào)節(jié)作用及其對(duì)aGVHD的影響 目的:蛋白酶體抑制劑硼替佐米移植后早期注射(移植后0-2天)可以預(yù)防aGVHD的發(fā)生。本研究旨在探討硼替佐米對(duì)宿主DC的調(diào)節(jié)作用及其對(duì)aGVHD的影響。揭示硼替佐米在移植后早期注射延緩aGVHD是通過(guò)其對(duì)DC成熟與功能的抑制作用實(shí)現(xiàn)的。 方法:體外在GM-CSF和IL-4條件下利用骨髓來(lái)源培養(yǎng)的DC,經(jīng)過(guò)不同濃度硼替佐米處理后,流式細(xì)胞術(shù)檢測(cè)協(xié)同共刺激分子CD40、CD80、CD86及MHC-II類抗原表達(dá)。用經(jīng)過(guò)不同濃度硼替佐米處理后的DC加入OVA肽段,與OT-I小鼠磁珠分離CD8+T細(xì)胞共育。胞內(nèi)染色觀察CD8+T細(xì)胞IL-2、IFN-γ和TNF-α表達(dá)情況,同時(shí)檢測(cè)上清中上述三種細(xì)胞因子的分泌量。EMSA檢測(cè)經(jīng)過(guò)不同濃度硼替佐米預(yù)處理的DC的NF-κB的入核活性。利用骨髓來(lái)源的培養(yǎng)DC經(jīng)過(guò)不同濃度硼替佐米預(yù)處理后的DC與異基因小鼠脾細(xì)胞共育,[3H]-TdR摻入法檢測(cè)硼替佐米處理后DC對(duì)異基因淋巴細(xì)胞增殖的影響。體內(nèi)利用硼替佐米預(yù)處理的宿主DC輸入到aGVHD小鼠體內(nèi),觀察小鼠生存狀態(tài)及生存率。 結(jié)果:小鼠骨髓來(lái)源的細(xì)胞在GM-CSF(10ng/mL)和IL-4(1 ng/mL)培養(yǎng)7天后CD11c陽(yáng)性率為85%以上。經(jīng)過(guò)不同濃度硼替佐米處理的DC在LPS刺激后,CD40、CD80、CD86及MHC-II類抗原表達(dá)下調(diào),呈現(xiàn)出明顯的劑量依賴性。經(jīng)過(guò)硼替佐米處理的DC與OT-I CD8+T細(xì)胞共育后檢測(cè)胞內(nèi)細(xì)胞因子流式圖顯示:CD8+TNF-α+T和CD8+INF-γ+T細(xì)胞數(shù)目逐漸減少,呈現(xiàn)出劑量依賴性。細(xì)胞上清細(xì)胞因子檢測(cè)顯示:CD8+T分泌TNF-α和INF-γ量逐漸減少,而IL-2的水平并無(wú)明顯變化。EMSA結(jié)果顯示,經(jīng)過(guò)硼替佐米預(yù)處理的imDC在LPS刺激條件下NF-κB入核量減少,并且呈現(xiàn)出濃度依賴性。經(jīng)過(guò)不同濃度硼替佐米處理的DC與異基因脾細(xì)胞進(jìn)行MLR實(shí)驗(yàn)結(jié)果顯示:DC在經(jīng)硼替佐米(2nM、10nM和50nM)處理后,其激活異基因T細(xì)胞增殖的能力隨著硼替佐米濃度的增高而逐漸減弱。經(jīng)過(guò)50nM硼替佐米處理后的1×10~6宿主DC輸入aGVHD模型小鼠后,其生存率要明顯高于未用硼替佐米處理而過(guò)繼輸入DC的aGVHD小鼠模型。 結(jié)論:經(jīng)過(guò)硼替佐米處理的DC其成熟受到抑制,繼而其抗原遞呈能力下調(diào),導(dǎo)致激活同種異基因淋巴細(xì)胞的增殖能力減弱。主要機(jī)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功能下調(diào),降低了異基因抗原的遞呈,從而預(yù)防了aGVHD的發(fā)生。 三、TLR4通路與IL-1β在硼替佐米延遲注射促進(jìn)aGVHD過(guò)程中的作用 目的:蛋白酶體抑制劑硼替佐米移植后延遲注射(移植后3天以后)可以促進(jìn)aGVHD的發(fā)生。本研究旨在探討TLR4通路與IL-1β在延遲注射硼替佐米促進(jìn)GVHD中的作用。 方法:體外系統(tǒng)模擬延遲注射與即時(shí)注射硼替佐米對(duì)DC和巨噬細(xì)胞細(xì)胞因子分泌的影響。培養(yǎng)骨髓來(lái)源的C57BL/6背景DC,于培養(yǎng)第7天LPS刺激,在LPS刺激前或刺激后加入硼替佐米。實(shí)驗(yàn)分四組:①延遲硼替佐米組:LPS(100ng/mL)刺激6 h后,加入不同濃度硼替佐米共育,24 h后檢測(cè)細(xì)胞因子濃度。②即時(shí)硼替佐米組:先加入不同濃度硼替佐米,6 h后再加入LPS (100ng/mL)刺激。③LPS對(duì)照組:直接加LPS刺激。④硼替佐米對(duì)照組:直接加不同濃度硼替佐米共育。利用上述方法處理的DC與異基因淋巴細(xì)胞進(jìn)行MLR反應(yīng),[3H]-TdR摻入法檢測(cè)硼替佐米處理后DC對(duì)T淋巴細(xì)胞增殖影響; ELISA法同時(shí)檢測(cè)上清中細(xì)胞因子TNF-α和IL-1β水平。 體內(nèi)采取三種不同的方式對(duì)aGVHD動(dòng)物模型進(jìn)行干預(yù),研究TLR4通路與硼替佐米延遲干預(yù)互作對(duì)aGVHD進(jìn)程的影響。 (1)阻斷TLR4通路對(duì)硼替佐米延遲注射促進(jìn)aGVHD的影響。用TLR4 KO小鼠做為受體,BALB/c為供體,單純輻照法(9.0Gy)建立小鼠GVHD模型,實(shí)驗(yàn)分即時(shí)注射硼替佐米組、延遲注射硼替佐米TLR4 KO組、延遲注射硼替佐米C57BL/6組、aGVHD C57BL/6模型對(duì)照組和aGVHD TLR4 KO模型對(duì)照組,觀察各組小鼠注射硼替佐米后的生存狀態(tài)及生存率。 (2)移植前預(yù)處理產(chǎn)生的不同LPS水平對(duì)硼替佐米延遲注射促進(jìn)aGVHD的影響。運(yùn)用氟達(dá)拉濱加小劑量照射(4Gy)預(yù)處理方式建立小鼠aGVHD模型,檢測(cè)不同時(shí)間小鼠體內(nèi)的LPS水平;觀察小鼠延遲注射硼替佐米生存率;同時(shí)根據(jù)體外模擬實(shí)驗(yàn)結(jié)果設(shè)上調(diào)體內(nèi)LPS濃度實(shí)驗(yàn),分GVHD模型對(duì)照組,延遲注射硼替佐米組,LPS+延遲注射硼替佐米組(建模第3天給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對(duì)照組(實(shí)驗(yàn)第3天給GVHD模型鼠注射LPS(5 mg/kg) 1次)。觀察小鼠生存狀態(tài)及生存率。 (3)阻斷炎性因子IL-1β對(duì)硼替佐米延遲注射促進(jìn)aGVHD的影響。大劑量輻照法建立小鼠aGVHD模型:選用SPF級(jí)接受8.5Gy致死劑量~(60)Co輻照的BALB/c小鼠作為受鼠,尾靜脈移植入C57BL/6小鼠1.0×10~7骨髓細(xì)胞和0.5×10~7脾細(xì)胞,建立小鼠aGVHD模型。骨髓移植后第1,3和5天取血,LAL法檢測(cè)小鼠LPS血清水平;同時(shí)檢測(cè)延遲注射硼替佐米組(骨髓移植后第3天注射,連續(xù)2天)、即時(shí)注射硼替佐米組(骨髓移植后連續(xù)注射2天)和GVHD模型組的Th1 (IFN-γ),Th2 (IL-4)、TNF-α與IL-1β血清水平。用上述方法建立小鼠aGVHD模型,實(shí)驗(yàn)分即時(shí)注射硼替佐米組、延遲注射硼替佐米組、阿那白滯素阻斷IL-1β組(骨髓移植后連續(xù)注射2天阿那白滯素)和阿那白滯素阻斷IL-1β+延遲注射硼替佐米組(骨髓移植后注射阿那白滯素阻滯IL-1β2天,第3天注射硼替佐米)。于延遲注射硼替佐米后小鼠瀕死時(shí)免疫組織化學(xué)法檢測(cè)各組小腸TNFR的表達(dá);ELISA法檢測(cè)血清細(xì)胞因子濃度;計(jì)算GVHD積分;觀察GVHD各組各個(gè)臟器(肝臟、皮膚、肺及小腸)HE染色病理組織切片及生存率。 結(jié)果:原代培養(yǎng)的DC在先LPS刺激6 h后與不同濃度硼替佐米的作用下比單用LPS刺激分泌的IL-1β顯著增高(P0.01);而TNF-α則無(wú)上述現(xiàn)象。我們用同樣的方法也檢測(cè)了巨噬細(xì)胞,未發(fā)現(xiàn)差異。經(jīng)延遲和即時(shí)處理的DC與同種異基因小鼠的脾細(xì)胞共育進(jìn)行MLR實(shí)驗(yàn),[3H]-TdR法檢測(cè)異結(jié)果顯示:較高濃度硼替佐米(50nM和10nM)延遲處理的DC其激活同種異基因淋巴細(xì)胞的能力要比LPS組和即時(shí)組都要強(qiáng)(P0.01)。延遲模擬組IL-4水平有顯著意義上升(P0.05)。 延遲給藥aGVHD小鼠血清細(xì)胞因子濃度結(jié)果顯示:延遲注射硼替佐米aGVHD組與aGVHD模型組或即時(shí)注射硼替佐米aGVHD組比,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,在延遲注射硼替佐米后不會(huì)立即死亡,且其生存率要顯著高于C57BL/6對(duì)照組和TLR4 KO模型對(duì)照組;而C57BL/6延遲注射組小鼠會(huì)立即死亡。 運(yùn)用氟達(dá)拉濱加小劑量輻照法與大劑量單獨(dú)輻照法建立的小鼠aGVHD模型相比,前者早期體內(nèi)LPS升高不明顯。不同預(yù)處理產(chǎn)生的LPS水平不同,在氟達(dá)拉濱加小劑量輻照組骨髓移植后,第1,3和5天的LPS水平分別為(0.17±0.02 )Eu/mL、(0.21±0.04 )Eu/mL和(0.23±0.05)Eu/mL;而大劑量輻照預(yù)處理組則為(0.27±0.02)Eu/mL、(0.47±0.02)Eu/mL和(0.69±0.04)Eu/mL。兩種方式比較結(jié)果:大劑量輻照組LPS濃度顯著高于氟達(dá)拉濱加小劑量輻照組(P0.01)。氟達(dá)拉濱加小劑量輻照組LPS水平相對(duì)穩(wěn)定,而大劑量輻照組則呈現(xiàn)出逐漸上升趨勢(shì)。運(yùn)用氟達(dá)拉濱與小劑量照射預(yù)處理方式建立的小鼠GVHD模型在延遲注射硼替佐米后生存期明顯比大劑量輻照組延長(zhǎng);在上調(diào)LPS實(shí)驗(yàn)中,LPS+延遲注射硼替佐米組與LPS對(duì)照組存在明顯差異,腸道腫脹,炎癥表現(xiàn)。 輻照法建立aGVHD模型后,用阿那白滯素阻斷IL-1β可以明顯延長(zhǎng)延遲注射硼替佐米小鼠的生存率。血清中IFN-γ與TNF-α下降,IL-4上升。組織病理變化、體重變化、體內(nèi)炎性因子變化均有差異。 結(jié)論:移植后期預(yù)處理造成腸道損傷,LPS激活TRL4通路,此時(shí)注射硼替佐米促進(jìn)了IL-1β的分泌,放大“炎性風(fēng)暴”,繼而過(guò)度激活宿主DC細(xì)胞及供者T細(xì)胞而促進(jìn)GVHD進(jìn)程。證明了TLR4信號(hào)通路激活是引發(fā)硼替佐米延遲注射導(dǎo)致GVHD相關(guān)性死亡的重要原因;并研究不同的預(yù)處理方式會(huì)導(dǎo)致機(jī)體產(chǎn)生不同水平的LPS,從而激活TLR4通路信號(hào)的強(qiáng)度不同,最終在延遲注射硼替佐米促進(jìn)GVHD相關(guān)性死亡中起到重要作用;IL-1β在延遲注射硼替佐米促進(jìn)aGVHD的進(jìn)程中起到了放大“炎性風(fēng)暴”的關(guān)鍵作用。
[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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392

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