發(fā)布時(shí)間：2018-03-18 01:05

  本文選題：非清髓性造血干細(xì)胞移植　切入點(diǎn)：供者細(xì)胞輸注　出處：《第四軍醫(yī)大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：背景造血干細(xì)胞移植(hematopoietic stem cell transplantation, HSCT)已經(jīng)成為治愈血液系統(tǒng)惡性疾病最有效的手段之一。但由于生物種系及個(gè)體間的主要組織相容性復(fù)合體(major histocompability complex, MHC)存在差異,同種異體移植時(shí)將產(chǎn)生雙向排斥反應(yīng),即移植物抗宿主(graft versus host, GVH)和宿主抗移植物(host versus graft, HVG)反應(yīng)。由于單倍體相合供受者間人類白細(xì)胞分化抗原(Human leukocyte antigen, HLA)差異顯著,移植排斥反應(yīng)重,移植前往往需要大劑量的放化療清除宿主免疫系統(tǒng)及腫瘤細(xì)胞,預(yù)防排斥、促進(jìn)植入,同時(shí)需去除供者干細(xì)胞中的T細(xì)胞以減輕移植物抗宿主病(graft-versus-host disease, GVHD),并且加大輸注的供者造血干細(xì)胞數(shù)量以促進(jìn)植入。非清髓性造血干細(xì)胞移植(nonmyeloablative hematopoietic stem cell transplantation, NST)由于移植前放化療強(qiáng)度降低,移植相關(guān)毒性減輕,越來(lái)越符合現(xiàn)代治療學(xué)的要求。然而非清髓性單倍體或不相合造血干細(xì)胞移植(mismatched hematopoieticstem cell transplantation, mis-HSCT)一方面預(yù)處理強(qiáng)度降低,另一面供受者主要組織相容性抗原不合,常導(dǎo)致移植物排斥,移植后供者細(xì)胞植入率低,甚至植入失敗。如何促進(jìn)植入一直是非清髓性單倍體相合造血干細(xì)胞移植的重點(diǎn)研究課題之一。如何在非清髓或減低毒性的(reduced intensity conditioning, RIC)前提下進(jìn)一步降低預(yù)處理強(qiáng)度,實(shí)現(xiàn)單倍體相合造血干細(xì)胞的植入,對(duì)于臨床移植具有重要意義。因其不僅保留了非清髓性單倍體相合造血干細(xì)胞移植的長(zhǎng)處,進(jìn)一步降低了移植相關(guān)毒性,解決了相合供者來(lái)源不足的問(wèn)題,同時(shí)弊棄了其短處,即移植排斥反應(yīng)大。目前已知,宿主體內(nèi)的T細(xì)胞是介導(dǎo)同種異體排斥反應(yīng)的主要免疫細(xì)胞,其清除或者誘導(dǎo)免疫無(wú)能對(duì)于促進(jìn)植入具有重要意義。去除或抑制供者同種異體反應(yīng)性T細(xì)胞可以明顯降低GVHD的發(fā)生率,表明同種異體反應(yīng)性細(xì)胞的去除對(duì)于減低異體排斥反應(yīng)確實(shí)可以減輕異體排斥。因而我們?cè)O(shè)想,通過(guò)去除受者體內(nèi)同種異體反應(yīng)性T的方法減輕宿主對(duì)供者細(xì)胞的排斥,促進(jìn)植入。阿糖胞苷為抗嘧啶類抗代謝藥,主要作用于S期的細(xì)胞,對(duì)于分裂活躍的細(xì)胞尤為敏感。體外實(shí)驗(yàn)證實(shí)免疫細(xì)胞接觸抗原64h時(shí)細(xì)胞毒作用達(dá)到峰值,我們通過(guò)移植前輸注供鼠細(xì)胞誘導(dǎo)宿主T細(xì)胞產(chǎn)生針對(duì)供鼠的同種異體反應(yīng),并在細(xì)胞免疫達(dá)到峰值之前利用阿糖胞苷將宿主體內(nèi)這種同種反應(yīng)性T細(xì)胞清除,這樣宿主體內(nèi)淋巴細(xì)胞池針對(duì)同一抗原的免疫能力相對(duì)耗竭,短時(shí)間內(nèi)接觸同一抗原,將產(chǎn)生免疫耐受,從而促進(jìn)供體細(xì)胞植入。 第一部分非清髓性單倍體相合骨髓移植小鼠模型的建立 目的建立小鼠非清髓性單倍體相合骨髓移植模型,為研究移植前輸注供體細(xì)胞誘導(dǎo)免疫耐受提供研究平臺(tái)。 方法實(shí)驗(yàn)分為實(shí)驗(yàn)組和對(duì)照組2組。以CB6F1雌性小鼠為受鼠,移植-1d予450cGy全身照射(TBI)后,隨機(jī)分為2組,實(shí)驗(yàn)組移植0d輸注C57BL/6雄性小鼠骨髓有核細(xì)胞5×107/只,對(duì)照組不予移植。監(jiān)測(cè)受鼠造血恢復(fù)、檢測(cè)供鼠Y染色體上性別決定基因(SRY),以及外周血供體細(xì)胞尤其CD3+細(xì)胞嵌合狀態(tài),同時(shí)觀察小鼠急性移植物抗宿主病(acute graft-versus-host disease, aGVHD)的發(fā)生情況。 結(jié)果對(duì)照組及實(shí)驗(yàn)組小鼠均未見(jiàn)小鼠死亡,各組小鼠均未見(jiàn)明顯aGVHD表現(xiàn),移植后30d內(nèi)白細(xì)胞基本恢復(fù)正常水平。實(shí)驗(yàn)組小鼠SRY基因在移植后14d、30d、60d時(shí)經(jīng)聚合酶鏈?zhǔn)椒磻?yīng)(polymerase chain reaction, PCR)檢測(cè),結(jié)果均陽(yáng)性,供鼠外周血淋巴細(xì)胞、單核細(xì)胞、粒細(xì)胞嵌合率移植后14d、30d、60d時(shí)分別為23.8%±4.6%、36.9%%±13.7%、19.4%±7.5% vs 49.9%±3.6%、53.2%±7.6%、54.4%±4.8% vs 67.6%±3.1%、51.6%±4.3%、56.9%±2.9%,其中CD3+細(xì)胞嵌合率分別為4.4%±4.6%、21.2%±6.4%、54.4%±4.1%。 結(jié)論450cGyTBI的非清髓性預(yù)處理方案,可以誘導(dǎo)受鼠免疫耐受、供體骨髓細(xì)胞植入,嵌合率處于中低水平混合嵌合狀態(tài)。 第二部分移植前輸注供體細(xì)胞誘導(dǎo)單倍體相合造血干細(xì)胞植入的實(shí)驗(yàn)研究 目的觀察移植前輸注供體細(xì)胞,能否誘導(dǎo)宿主免疫耐受、促進(jìn)供體骨髓細(xì)胞植入。 方法實(shí)驗(yàn)分為Ara-C+TBI+BMT, SC+Ara-C+TBI+BMT, BMC+ Ara-C+ TBI+BMT 3組,以CB6F1雌性小鼠為受鼠,C57BL/6雄性小鼠為供鼠,移植前-3d經(jīng)尾靜脈輸注供鼠脾細(xì)胞或骨髓細(xì)胞3×107,輸注細(xì)胞后24h和48h分別通過(guò)腹腔注射阿糖胞苷0.015g/d,-1d時(shí)予450cGy全身照射(TBI),移植當(dāng)天輸注供鼠骨髓有核細(xì)胞5×107/只。監(jiān)測(cè)受鼠白細(xì)胞恢復(fù)、外周血供鼠CD3+細(xì)胞嵌合狀態(tài)及小鼠存活、GVHD的發(fā)生。 結(jié)果各組分別有兩只小鼠死亡,并表現(xiàn)不同程度GVHD。移植前輸注脾細(xì)胞組和對(duì)照組白細(xì)胞恢復(fù)中位時(shí)間相當(dāng),為17d(12d~37d),輸注骨髓細(xì)胞組恢復(fù)時(shí)間稍慢,中位時(shí)間為22d(22d~22d)。移植前輸注供體脾細(xì)胞組及對(duì)照組供鼠CD3+細(xì)胞嵌合率相當(dāng),均達(dá)到完全供者型嵌合,+30d時(shí)移植前輸注脾細(xì)胞組供鼠源CD3+細(xì)胞比例達(dá)93.5%±4.8%,明顯高于輸注骨髓細(xì)胞組(P0.05),+60d時(shí),輸注骨髓細(xì)胞組供體細(xì)胞嵌合率上升為87.2%±7.4%,與輸注脾細(xì)胞組嵌合水平相當(dāng)(91.7%±4.0%,P0.05),各組小鼠均出現(xiàn)不同程度的GVHD。 結(jié)論移植前輸注脾細(xì)胞可在一定程度上促進(jìn)供體造血干細(xì)胞的早期植入。
[Abstract]:The background of hematopoietic stem cell transplantation (hematopoietic stem cell transplantation, HSCT) has become one of the most effective cure of malignant hematological diseases means. But due to the biological tissue and major histocompatibility complex between individuals (major histocompability, complex, MHC) differences in allograft rejection will have a two-way, or graft the host (graft versus host, GVH) and graft versus host (host versus, graft, HVG) reaction. Because haploidentical donor human leukocyte differentiation antigen (Human leukocyte, antigen, HLA) had significant difference, transplant rejection, often require high dose chemotherapy before transplantation of the host immune system and tumor removal cells, preventing rejection, promoting implantation, and removal of donor stem cells in T cells to reduce graft-versus-host disease (graft-versus-host disease, GVHD), and Increase the infusion of donor hematopoietic stem cell implantation. In order to promote the number of non myeloablative hematopoietic stem cell transplantation (nonmyeloablative hematopoietic stem cell transplantation, NST) before transplantation due to chemotherapy intensity decrease, reduce transplant related toxicity, more in line with the requirements of modern therapeutics. However non myeloablative hematopoietic stem haploid or not cell transplantation (mismatched hematopoieticstem cell transplantation, mis-HSCT) pretreatment reduces the intensity of the one hand, on the other side of the main organization for recipients of MHC incompatibility, often leads to graft rejection, low rate of donor cell engraftment after transplantation, or implant failure. How to promote implantation has been the focus of study of nonmyeloablative haploidentical hematopoietic stem cell transplantation. In non myeloablative or reduced toxicity (reduced intensity, conditioning, RIC) under the premise of further reducing pretreatment The strength of haploidentical hematopoietic stem cell implantation, has important significance for clinical transplantation. Because it not only retains the non myeloablative allogeneic hematopoietic stem cell transplantation strengths, further reducing transplant related toxicity, solve the matched donor shortage problem at the same time, abandoned the disadvantages of its weaknesses, namely the transplant rejection the reaction. It is now known that host T cells are the main immune cell mediated allograft rejection, the scavenging or induce immune anergy and plays an important role in promoting or inhibiting implantation. Removal of donor alloreactive T cells can significantly reduce the incidence of GVHD, suggesting that removal of alloreactive cells for reduce allograft rejection can indeed reduce rejection. So we assume that, by removing the body by the method of alloreactive T donor cells to reduce the host The rejection, promote implantation. Cytarabine for anti pyrimidine antimetabolites, a major role in the S phase of the cell, particularly sensitive to mitotically active cells. In vitro immune cells with the antigen 64H cytotoxic effect reached the peak, we through the pretransplant infusion of donor cells induced by host T cells for the same allogeneic response of donor rats, and reached the peak in cellular immunity before using cytarabine will host the alloreactive T cell depletion, immunity against the same antigen that host lymphocyte pool is depleted, contact time will produce the same antigen, immune tolerance, thereby promoting engraftment.
The first part of a mouse model of non myeloablative haploid allograft bone marrow transplantation
Objective to establish a model of non myelohaploid allograft bone marrow transplantation in mice, and to provide a research platform for the study of immune tolerance induced by donor cells before transplantation.
Methods the experiments were divided into experimental group and control group. 2 groups of female CB6F1 mice as the recipients in transplantation -1d to 450cGy irradiation (TBI), were randomly divided into 2 groups, experimental group was transplanted with 0d infusion of C57BL/6 male mice bone marrow nucleated cells of 5 * 107/, the control group was not monitored by murine hematopoietic transplantation. Recovery, detection for the mouse Y chromosome sex determining gene (SRY), and peripheral blood donor cells especially CD3+ cell chimerism, and observe the mice of acute graft-versus-host disease (acute graft-versus-host, disease, aGVHD) were observed.
The results of the control group and the experimental group mice showed no death of the mice, the mice showed no obvious aGVHD expression, white blood cell 30d returned to normal levels after transplantation. The experimental group of mice after transplantation of SRY gene in 14d, 30d, 60d by polymerase chain reaction (polymerase chain reaction, PCR) detection, the results were positive for rats peripheral blood lymphocytes, monocytes, myeloid chimerism after transplantation of 14d, 30d, 60d were 23.8% + 4.6%, 36.9%% + 13.7%, 19.4% + 7.5% vs 49.9% + 3.6%, 53.2% + 7.6%, 54.4% + 4.8% vs 67.6% + 3.1%, 51.6% + 4.3%, 56.9% + 2.9%, including CD3+ cell chimerism were 4.4% + 4.6%, 21.2% + 6.4%, 54.4% + 4.1%.
Conclusion the non myeloablative preconditioning scheme of 450cGyTBI can induce immune tolerance, bone marrow cells implantation and chimerism in low and middle level.
Experimental study of haploid allogeneic stem cell implantation induced by donor cells in second parts before transplantation
Objective To observe whether donor cells were transfused before transplantation to induce host immune tolerance and to promote donor bone marrow cells implantation.
Methods the experiment was divided into Ara-C+TBI+BMT, SC+Ara-C+TBI+BMT, BMC+ Ara-C+, TBI+BMT 3 groups, CB6F1 female mice as recipients were male C57BL/6 mice as donors and -3d before transplantation via tail vein infusion of donor spleen cells or bone marrow cells 3 * 107 cells after infusion of 24h and 48h respectively through abdominal cavity injection of cytarabine 0.015g/d -1d, with 450cGy total body irradiation (TBI), on the day of transplantation by infusion of bone marrow nucleated cells of 5 * 107/. Recipients were monitored the recovery of leukocytes, peripheral blood donor mice CD3+ cell chimerism and survival, the occurrence of GVHD.
The results of each group were two mice died, and showed different degrees of GVHD. before transplantation group and control group injection of spleen cells of white blood cell recovery in time is equivalent to 17D (12d~37d), infusion of bone marrow cells group the recovery time is slower, the median time to 22d (22d~22d) before transplantation. Infusion of donor spleen cells group and the control group of donor CD3+ cell chimerism, achieved complete donor chimerism, +30d infusion of spleen cells group before transplantation for murine CD3+ cell ratio was 93.5% + 4.8%, was significantly higher than that of infusion of bone marrow cell group (P0.05), +60d, infusion of bone marrow cells for somatic cell chimerism rate rise 87.2% + 7.4%, and lose chimeric horizontal injection spleen cells group (91.7% + 4%, P0.05), the mice showed varying degrees of GVHD.
Conclusion the injection of splenocytes before transplantation can promote the early implantation of donor hematopoietic stem cells to a certain extent.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392

【共引文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王春燕;譚獲;郭坤元;;異基因性NK細(xì)胞在小鼠體內(nèi)抗白血病作用[J];吉林大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2007年04期

相關(guān)博士學(xué)位論文 前5條

1 王易;小鼠臍血移植模型的建立及角質(zhì)細(xì)胞生長(zhǎng)因子在移植后免疫重建中作用機(jī)制研究[D];蘇州大學(xué);2011年

2 王春燕;供者NK細(xì)胞在小鼠MHC半相合骨髓移植的作用[D];第一軍醫(yī)大學(xué);2004年

3 李曉峰;單倍體相合細(xì)胞移植治療小鼠H22實(shí)體瘤的實(shí)驗(yàn)研究[D];福建醫(yī)科大學(xué);2006年

4 楊明珍;雷帕霉素誘導(dǎo)的CD4~+CD25~+T調(diào)節(jié)細(xì)胞對(duì)單倍型相合造血干細(xì)胞移植的影響及HLA-Cw位點(diǎn)在單倍型相合造血干細(xì)胞移植中的作用[D];蘇州大學(xué);2007年

5 陳廣華;供者自然殺傷細(xì)胞輸注及IL-2、IL-15治療在異基因及單倍型相合造血干細(xì)胞移植中作用的實(shí)驗(yàn)研究[D];蘇州大學(xué);2009年

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