本文選題：造血干細(xì)胞移植　切入點：預(yù)處理　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2011年博士論文

【摘要】：研究目的:異基因造血干細(xì)胞移植(allo-HSCT)是目前治療惡性血液系統(tǒng)疾病、新陳代謝疾病、免疫系統(tǒng)缺陷、自身免疫系統(tǒng)疾病以及實體腫瘤等多種疾病的有效方法。長期以來一致認(rèn)為:足夠強度的預(yù)處理(髓系和淋巴系的徹底清除-清髓性移植;淋巴系的徹底清除及一定程度的髓系清除-非清髓移植)及骨髓騰空是供體細(xì)胞穩(wěn)定植入及誘導(dǎo)移植物抗白血病效應(yīng)(GVL)的基礎(chǔ)和必要條件。然而預(yù)處理相關(guān)的毒性反應(yīng)及移植后GVHD、排斥、感染等并發(fā)癥嚴(yán)重限制了allo-HSCT的療效。與傳統(tǒng)的清髓方案相比,非清髓等減毒方案雖然顯著減輕了預(yù)處理相關(guān)的并發(fā)癥,但預(yù)處理本身所包含的放化療、移植后免疫抑制劑的應(yīng)用,以及免疫功能低下所致的細(xì)菌、真菌及病毒等嚴(yán)重感染并發(fā)癥仍嚴(yán)重威脅著病人的生命和健康。因此,如何從根本上減輕移植相關(guān)并發(fā)癥及病死率,進(jìn)一步提高移植療效是臨床亟需解決的問題。 本課題旨在既往清髓和非清髓移植和HLA半相合移植研究的基礎(chǔ)上,試圖通過最大限度的減小或去除預(yù)處理,但調(diào)整供體細(xì)胞數(shù)量等條件,達(dá)到供體細(xì)胞穩(wěn)定植入及預(yù)防GVHD。本課題擬從另一角度研究異基因供體細(xì)胞植入及免疫耐受和GVHD相關(guān)科學(xué)問題及機制研究,同時探討無預(yù)處理條件下進(jìn)行allo- HSCT的可行性及安全性,為非惡性疾病、無預(yù)處理條件下的allo-HSCT的臨床應(yīng)用提供實驗依據(jù)。 研究內(nèi)容:首先建立FCM檢測小鼠供體大嵌合和Real-time PCR檢測小鼠供體微嵌合的方法,并評價方法的有效性,其次研究不同TBI強度(8GY~1GY)對H-2半相合移植小鼠供體植入和GVHD的影響規(guī)律,進(jìn)一步研究不同供體細(xì)胞輸注數(shù)量(1×10~7～9×10~7)對H-2半相合移植小鼠供體植入和GVHD的影響規(guī)律。然后研究在無預(yù)處理條件下,輸注不同數(shù)量的H-2半相合供體細(xì)胞組小鼠供體植入情況與GVHD情況,探討無預(yù)處理條件下H-2半相合移植的可行性及安全性。最后通過無預(yù)處理條件下供體細(xì)胞分布、淋巴細(xì)胞亞群變化及細(xì)胞因子變化等研究探討無預(yù)處理條件下H-2半相合供體細(xì)胞輸注誘導(dǎo)供體細(xì)胞穩(wěn)定植入的可能機制。 研究方法:通過檢測移植后受鼠H-2表型變化建立FCM檢測供體大嵌合的方法;建立Real-time PCR法檢測SRY基因表達(dá)率即供體微嵌合的方法:首先根據(jù)小鼠β-actin基因及sry基因序列設(shè)計引物探針、進(jìn)行PCR擴增,再將電泳產(chǎn)物回收構(gòu)建質(zhì)粒,測序正確后建立標(biāo)準(zhǔn)曲線,然后進(jìn)行實時熒光定量PCR擴增并優(yōu)化擴增條件,最后以雄性小鼠DNA標(biāo)本為陽性對照,雌性小鼠為陰性對照,檢驗Real-time PCR法的敏感性、特異性以及可重復(fù)性。以TBI 6GY與2GY條件下H-2半相合骨髓移植小鼠模型為例,通過檢測兩組小鼠移植后DC驗證FCM法聯(lián)合Real-time PCR法作為DC定量檢測方法的有效性; 分別以親代和子代小鼠作為供鼠與受鼠,TBI作為預(yù)處理,進(jìn)行H-2半相合造血干細(xì)胞移植研究。實驗組根據(jù)不同TBI強度(8GY、6GY、4GY、2GY、1GY)分為5組,分別輸注動員后的供鼠脾單個核細(xì)胞1×10~7/只,對照組在TBI后輸注等量生理鹽水。比較不同TBI強度組移植后小鼠供體植入情況、GVHD發(fā)生率與病死率、生存情況的差異;實驗組在相同TBI條件下,再根據(jù)不同供體細(xì)胞輸注數(shù)量(1×10~7、3×10~7、6×10~7、9×10~7)分為A組～D組。比較不同細(xì)胞數(shù)量組小鼠移植后供體植入及GVHD情況的差異;在無預(yù)處理條件下根據(jù)不同供體細(xì)胞輸注數(shù)量(1×10~7、3×10~7、6×10~7、9×10~7、12×10~7、15×10~7)分為6組,比較各組小鼠輸注H-2半相合供體細(xì)胞后供體植入及GVHD的差異;檢測受鼠在無預(yù)處理條件下輸注H-2半相合供體細(xì)胞后淋巴細(xì)胞亞群變化、細(xì)胞因子變化、供體細(xì)胞組織分布以及細(xì)胞系中DC情況。 研究結(jié)果:成功建立了小鼠供體嵌合率定量檢測方法;6GY組小鼠在移植2w后DC90%,利用FCM法檢測精確度較高,Real-time PCR法檢測誤差較大;2GY組小鼠在移植1月后DC1% ,利用Real-time PCR法檢測良好,FCM法靈敏度較低無法檢測。兩種方法結(jié)合運用,可適于移植后不同供體嵌合狀態(tài)的檢測。 在相同的供體細(xì)胞數(shù)量1×10~7的移植條件下,TBI 8GY組小鼠移植后2w即獲得完全供體植入(CC),并出現(xiàn)典型的GVHD體征,GVHD發(fā)生率100%,在移植后18d～25d全部死亡;6GY組小鼠在移植后6w也獲得了CC,3/8小鼠出現(xiàn)了輕度的GVHD,全部小鼠存活時間大于3個月;4GY組小鼠移植后僅獲得了MC,1/8小鼠出現(xiàn)輕度GVHD,小鼠均長期存活;2GY組與1GY組小鼠移植后1w獲得了低比例的MC,然后轉(zhuǎn)為微嵌合狀態(tài),供體細(xì)胞分別在10w與8w內(nèi)完全消失,無GVHD的發(fā)生。 在TBI-6GY條件下,供體細(xì)胞數(shù)量3×10~7組、6×10~7組及9×10~7組小鼠移植后MC轉(zhuǎn)為CC的時間分別是3w、2.5w、2w,且均出現(xiàn)了典型的GVHD體征,GVHD發(fā)病率100%,病死率分別為75%～100%;在TBI-4GY條件下供體細(xì)胞數(shù)量3×10~7組、6×10~7組及9×10~7組小鼠移植后均獲得了CC,GVHD死亡率分別為25%、50%、100%;在TBI-2GY/1GY條件下供體細(xì)胞數(shù)量3×10~7組小鼠移植后均獲得了MC及短暫的供體微嵌合狀態(tài),未發(fā)生GVHD;TBI-2GY條件下6×10~7組及9×10~7組小鼠移植后均獲得CC,出現(xiàn)典型了GVHD癥狀,GVHD發(fā)生率為62.5%與100%,GVHD病死率分別為25%與50%;TBI-1GY條件下6×10~7組及9×10~7組小鼠移植后均獲得CC,出現(xiàn)輕度GVHD癥狀,GVHD發(fā)病率分別為25%與62.5%,小鼠均長期存活。 無預(yù)處理條件下,1×10~7組、3×10~7組以及6×10~7組小鼠在輸注H-2半相合供體細(xì)胞后獲得一過性的供體細(xì)胞微嵌合,供體細(xì)胞在受鼠體內(nèi)存在的中位時間分別是5w、7w及9w,未發(fā)生GVHD。9×10~7組5/8小鼠在輸注細(xì)胞后8w(5w～12w)獲得CC,且未見明顯的GVHD體征。12×10~7組及15×10~7組小鼠在移植后全部獲得了CC,MC轉(zhuǎn)為CC的中位時間分別是6w與5w,GVHD發(fā)生率分別為0和25%,小鼠存活時間均大于6個月。 無預(yù)處理條件下輸注半相合供體細(xì)胞后受鼠造血及淋巴細(xì)胞亞群短暫抑制但可逐漸恢復(fù)正常;無預(yù)處理組受鼠血清中TH1細(xì)胞因子(IFN-γ、IL-2)及炎性因子TNF-α表達(dá)較GVHD組降低,TH2細(xì)胞因子(IL-4、IL-10、IL-6)較對照組明顯升高;MLR實驗提示6×10~7組和12×10~7組小鼠在移植后對供體細(xì)胞的反應(yīng)性較正常小鼠下降,而對第三方供體細(xì)胞的反應(yīng)性與正常鼠無差異;組織中及亞群中供體嵌合率檢測結(jié)果表明無預(yù)處理條件下12×10~7組小鼠移植后獲得的供體嵌合體為多系嵌合體,供體細(xì)胞植入特點及在各免疫器官的分布與預(yù)處理組類似。 研究結(jié)論:本研究成功建立了FCM聯(lián)合Real-time PCR法定量檢測小鼠供體嵌合率的方法;闡明了在H-2半相合移植模型中TBI強度與供體細(xì)胞輸注數(shù)量均是影響小鼠供體植入與GVHD的重要因素;成功建立了在無預(yù)處理條件下的小鼠H-2半相合移植模型;初步確定了無預(yù)處理方案中保障移植成功而無GVHD的適宜供體細(xì)胞數(shù)量;揭示無預(yù)處理方案可能通過形成組織中混合嵌合體(MC)、調(diào)節(jié)TH1/TH2平衡、調(diào)節(jié)性T淋巴細(xì)胞等機制誘導(dǎo)供體細(xì)胞穩(wěn)定植入和免疫耐受。此模型的建立及相關(guān)機制研究為無預(yù)處理的半相合移植方案在臨床的應(yīng)用提供動物實驗基礎(chǔ)和理論依據(jù)。
[Abstract]:Research purposes : Allogeneic hematopoietic stem cell transplantation ( allogeneic hematopoietic stem cell transplantation ) is an effective method for the treatment of malignant blood system diseases , metabolic diseases , immune system defects , autoimmune diseases and solid tumors .

The purpose of this study was to study the feasibility and safety of allogeneic donor cell implantation and immune tolerance and GVHD , and to explore the feasibility and safety of allogeneic donor cell implantation and immune tolerance and GVHD .

In this paper , the effects of donor implantation and GVHD in mice with H - 2 half - phase transplantation were studied by means of FCM . The effects of different donor - donor infusion numbers ( 8GY - 1GY ) on donor implantation and GVHD in H - 2 half - phase grafting mice were studied . The possible mechanism of donor cell distribution , lymphocyte subsets and cytokine changes was studied under the condition of no pretreatment .

Methods : To establish a method for the detection of donor microchimerism by detecting H - 2 phenotype changes in mice after transplantation . A method for detecting SRY gene expression rate , i.e . donor microchimerism , was established by establishing a real - time PCR method . The PCR amplification was carried out according to the mouse 尾 - actin gene and the sry gene sequence . The sensitivity , specificity and repeatability of the real - time PCR method were determined .

The experimental group was divided into 6 groups according to different donor cell infusion numbers ( 1 脳 10 ~ 7 , 3 脳 10 ~ 7 , 6 脳 10 ~ 7 , 9 脳 10 ~ 7 , 12 脳 10 ~ 7 , 15 脳 10 ~ 7 ) . The experimental groups were divided into 6 groups according to different donor cell infusion numbers ( 1 脳 10 ~ 7 , 3 脳 10 ~ 7 , 6 脳 10 ~ 7 , 9 脳 10 ~ 7 , 12 脳 10 ~ 7 , 15 脳 10 ~ 7 ) . The experimental groups were divided into 6 groups according to the number of different donor cells ( 1 脳 10 ~ 7 , 3 脳 10 ~ 7 , 6 脳 10 ~ 7 , 9 脳 10 ~ 7 , 12 脳 10 ~ 7 , 15 脳 10 ~ 7 ) .

Results : The method of quantitative determination of mouse donor chimerism was successfully established ; 6GY group of mice were DC90 % after 2w transplantation , the accuracy was high with FCM method , the detection accuracy was high with the method of real - time PCR , and the sensitivity of the 2GY group was less than that of the method of real - time PCR , and the sensitivity of FCM method was low . The two methods were combined and applied , which could be suitable for the detection of the chimeric state of different donor after transplantation .

At the same donor cell number of 1 脳 10 ~ 7 , the total donor implant ( CC ) was obtained after transplantation of 8GY group of mice . The incidence of GVHD was 100 % . After transplantation , the mice showed mild GVHD . All the mice survived longer than 3 months . After transplantation , only MC and 1 / 8 mice received low proportion of MC , then the mice survived . The donor cells disappeared completely within 10w and 8w , and no GVHD occurred .

There were 3 脳 10 ~ 7 groups of donor cells , 6 脳 10 ~ 7 groups and 9 脳 10 ~ 7 groups of mice after transplantation . The rates of GVHD and GVHD were 25 % , 50 % and 100 % , respectively .

There were no obvious GVHD signs . The median time of CC and MC to CC was 0 and 25 % in 12 脳 10 ~ 7 groups and 15 脳 10 ~ 7 groups , respectively , and the survival time of mice was more than 6 months .

The results showed that the donor chimeras obtained after transplantation of 12 脳 10 ~ 7 groups and 12 脳 10 ~ 7 groups was similar to that of normal mice after transplantation . The donor chimeras obtained after transplantation of mice with no pre - treatment was 6 脳 10 ~ 7 and 12 脳 10 ~ 7 groups .

Conclusion : This study successfully established a method for detecting the donor chimerism rate in mice by means of FCM combined with Real - time PCR . It has been shown that both the intensity and the number of donor cells in the H - 2 half - phase grafting model are important factors affecting donor implantation and GVHD in mice . The establishment of a pretreatment protocol and the mechanism of non - pretreatment can induce the stable implantation and immune tolerance of donor cells . The establishment of this model and the related mechanism are used to provide animal experimental basis and theoretical basis for the clinical application of the non - pretreatment half - phase grafting scheme .

【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R392.1

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