本文選題：臍血移植 + 植入前綜合征��； 參考：《安徽醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的植入前綜合征(PES)是一種高發(fā)于非血緣臍血移植(UCBT)后在中性粒細(xì)胞植入之前,以發(fā)熱、皮疹、腹瀉等為主要臨床表現(xiàn)的一種免疫反應(yīng)。發(fā)生重癥PES的患者,移植相關(guān)死亡率明顯增高。本研究通過對(duì)UCBT后發(fā)生PES患者外周血進(jìn)行供受者基因嵌合狀態(tài)檢測(cè),了解發(fā)生PES時(shí)細(xì)胞的來源;根據(jù)血清細(xì)胞因子檢測(cè)結(jié)果發(fā)現(xiàn)可溶性ST2(sST2)在PES發(fā)生時(shí)明顯升高,選擇新鮮臍血和外周血干細(xì)胞,采用IL-33和sST2進(jìn)行體外刺激,探索發(fā)生PES的效應(yīng)細(xì)胞,為PES發(fā)病機(jī)制的研究奠定基礎(chǔ)。方法本研究分為兩部分。第一部分:UCBT后發(fā)生PES的細(xì)胞來源的研究。根據(jù)本中心先前的研究發(fā)現(xiàn)UCBT后PES發(fā)生的中位時(shí)間為UCBT后第7天(+7d),選擇移植患者+7d的外周血標(biāo)本,采用短串聯(lián)重復(fù)序列聚合酶鏈?zhǔn)椒磻?yīng)(STR-PCR)技術(shù)檢測(cè)發(fā)生PES組和未發(fā)生PES組患者供受者嵌合狀態(tài),分析發(fā)生PES的細(xì)胞來源。第二部分:UCBT后發(fā)生PES的效應(yīng)細(xì)胞的探討。一、UCBT后PES發(fā)生時(shí)各種免疫細(xì)胞及其亞群和相關(guān)細(xì)胞因子的檢測(cè)。檢測(cè)PES發(fā)生時(shí)的T、B、NK和單核細(xì)胞及其亞群的變化;檢測(cè)患者移植前、移植當(dāng)天、PES發(fā)生時(shí)不同時(shí)間點(diǎn)的外周血的白細(xì)胞數(shù),血漿C-反應(yīng)蛋白和血清中相關(guān)細(xì)胞因子的變化。二、不同來源的移植物體外刺激實(shí)驗(yàn):sST2在PES發(fā)生時(shí)顯著增高,而ST2是IL-33的孤兒受體,擬從IL-33/ST2信號(hào)通路來推證PES的效應(yīng)細(xì)胞。應(yīng)用IL-33,sST2刺激新鮮臍血、外周血干細(xì)胞(動(dòng)員采集的同胞供者),每種類型的細(xì)胞分4組:實(shí)驗(yàn)組:A.IL-33+sST2組;對(duì)照組:B.sST2組;C.IL-33組;D.空白組。經(jīng)體外培養(yǎng)0、12、24和72小時(shí)后,細(xì)胞計(jì)數(shù)板計(jì)算刺激前后細(xì)胞總數(shù),流式細(xì)胞術(shù)檢測(cè)刺激前后細(xì)胞亞群及ST2受體表達(dá)情況。結(jié)果第一部分UCBT后發(fā)生PES的細(xì)胞來源的研究STR-PCR檢測(cè)臍血及受者移植前后的標(biāo)本比對(duì)供受者的嵌合狀態(tài),結(jié)果顯示:UCBT+7d供者的嵌合度發(fā)生PES組明顯高于未發(fā)生PES組,分別為 70.58±22.86%與37.18±18.96%,兩組之間有顯著的統(tǒng)計(jì)學(xué)差異,P0.0001。第二部分UCBT后發(fā)生PES的效應(yīng)細(xì)胞的探討一、UCBT后發(fā)生PES時(shí)各種免疫細(xì)胞及其亞群和相關(guān)細(xì)胞因子的檢測(cè)1.白細(xì)胞的變化PES組與無PES組在移植前、移植當(dāng)天、PES發(fā)生時(shí)的白細(xì)胞計(jì)數(shù)組間無統(tǒng)計(jì)學(xué)差異,P0.05。2.PES發(fā)生時(shí)T、B、NK和單核細(xì)胞的細(xì)胞亞群檢測(cè)結(jié)果:CD45+CD3+T淋巴細(xì)胞、CD45+CD3-CD19+B淋巴細(xì)胞、CD45+CD3-CD56+NK細(xì)胞、CD45+CD3-CD14+單核細(xì)胞分別占90%,0.3%,2.1%和0.2%。PES組與無PES組的T、B、NK和單核細(xì)胞的各細(xì)胞亞群比例無明顯統(tǒng)計(jì)學(xué)差異,P0.05。3.C-反應(yīng)蛋白的變化在移植前、移植當(dāng)天、PES發(fā)生時(shí),PES組和無PES組兩組之間無顯著統(tǒng)計(jì)學(xué)差異,P0.05。4.血清細(xì)胞因子的變化發(fā)生PES時(shí)細(xì)胞因子IL-6、MCP-1和sST2在PES組明顯高于無PES組(P0.05);PES組自身比較患者發(fā)生PES時(shí)也顯著高于移植前、移植當(dāng)天的水平(P0.05)。PES患者治療有效者細(xì)胞因子IL-6和MCP-1明顯下降并恢復(fù)至發(fā)生前水平(P0.01),而sST2與PES發(fā)生時(shí)相比雖有所下降但無統(tǒng)計(jì)學(xué)差異(P0.05)。發(fā)生PES時(shí)IFN-γ、IL-2、IL-1Rα、IL-4、IL-8、IL-17A、IL-33、MIP-1α、MIP-1β和TNF-α表達(dá)水平兩組相近無統(tǒng)計(jì)學(xué)差異(P0.05),同時(shí)PES組患者在不同的時(shí)間點(diǎn),自身對(duì)比其表達(dá)水平相近無統(tǒng)計(jì)學(xué)差異(P0.05)。二、不同來源的移植物體外刺激實(shí)驗(yàn)1.細(xì)胞數(shù)量的變化:新鮮臍血和外周血干細(xì)胞經(jīng)IL-33、sST2刺激培養(yǎng)后,各組刺激前后細(xì)胞數(shù)量無明顯增加(P0.05),兩種來源不同的細(xì)胞刺激培養(yǎng)12h后均出現(xiàn)多形性變化,此后隨培養(yǎng)時(shí)間延長形態(tài)變化不顯著。2.T、B、NK和單核細(xì)胞各亞群的變化:新鮮臍血經(jīng)Ficoll提取單個(gè)核細(xì)胞后,臍血提取的單個(gè)核細(xì)胞比例分別為T淋巴細(xì)胞30-50%、B淋巴細(xì)胞1-3%,NK細(xì)胞5-8%,單核細(xì)胞5-13%。外周血干細(xì)胞中T淋巴細(xì)胞20-35%、B淋巴細(xì)胞2-5%,NK細(xì)胞3-9%,單核細(xì)胞18-32%。經(jīng)刺激培養(yǎng)12、24和72h后,四組的T、B、NK和單核細(xì)胞亞群比例無明顯差異(P0.05),隨著培養(yǎng)時(shí)間的延長, 有少量細(xì)胞凋亡,實(shí)驗(yàn)各組細(xì)胞不同培養(yǎng)時(shí)間點(diǎn)的細(xì)胞亞群比例也無明顯統(tǒng)計(jì)學(xué)差異(P0.05)。3.T、B、NK和單核細(xì)胞ST2受體表達(dá)情況:新鮮臍血和外周血干細(xì)胞中ST2受體表達(dá)相似,單核細(xì)胞上ST2受體表達(dá)量高達(dá)53-90%,其次是B淋巴細(xì)胞亞群約為30-60%,T淋巴細(xì)胞及NK細(xì)胞亞群表達(dá)量低,分別為2-5%和3-10%。而四組的各細(xì)胞亞群ST2受體表達(dá)無明顯變化(P0.05),實(shí)驗(yàn)各組細(xì)胞不同培養(yǎng)時(shí)間點(diǎn)的各細(xì)胞亞群ST2表達(dá)也無顯著變化(P0.05)。結(jié)論1.UCBT后PES是供者開始植入時(shí)引發(fā)多種細(xì)胞因子參與的免疫反應(yīng),其效應(yīng)細(xì)胞推測(cè)來源于供者,供者的早期嵌合是PES發(fā)生的高危因素。2.血清中單核細(xì)胞相關(guān)的趨化因子(MCP-1)在PES發(fā)生時(shí)明顯升高,而淋巴細(xì)胞相關(guān)的較特異的細(xì)胞因子(IL-2、IL-4、IL-8、IL-17A)在PES發(fā)生時(shí)無顯著變化,PES的臨床表現(xiàn)主要在皮膚、腸道等組織中,推斷組織中的單核巨噬細(xì)胞可能是PES的主要效應(yīng)細(xì)胞。3.加入IL-33和sST2進(jìn)行單純的體外培養(yǎng),未見到免疫細(xì)胞的增殖和ST2受體表達(dá)的增高,但不能確定細(xì)胞因子水平是否發(fā)生變化,需進(jìn)一步檢測(cè)細(xì)胞培養(yǎng)液中細(xì)胞因子的變化,觀察IL-33/ST2信號(hào)通路及sST2是否對(duì)下游細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)存在影響,從而引起細(xì)胞因子的分泌。同時(shí)本實(shí)驗(yàn)也需要加測(cè)各免疫細(xì)胞活化的指標(biāo),如HLA-DR,CD27,CCR等等,為揭示PES的發(fā)生機(jī)制提供依據(jù)。
[Abstract]:Objective preimplantation syndrome (PES) is an immune response to the main clinical manifestations of fever, rash, and diarrhoea before neutrophils implantation after high incidence of non related umbilical cord blood transplantation (UCBT). Patients with severe PES have a significant increase in transplant related mortality. This study was conducted through the study of peripheral blood in patients with PES after UCBT. Detection of gene chimerism to understand the source of cells when PES occurs. According to the results of serum cytokine detection, it is found that soluble ST2 (sST2) is obviously elevated when PES occurs, select fresh umbilical blood and peripheral blood stem cells, use IL-33 and sST2 to stimulate in vitro, explore the effect cells of PES, and lay the foundation for the study of pathogenesis of PES. Methods this study was divided into two parts. Part one: a study of the cell origin of PES after UCBT. According to previous studies in this center, the median time of PES after UCBT was found to be seventh days after UCBT (+7d), and the peripheral blood samples of the transplanted +7d were selected and the PES group was detected by the short tandem repeat polymerase chain reaction (STR-PCR) technique. PES group of donor chimerism, analysis of PES cell origin. Second part: the effect cells of PES after UCBT. 1, detection of various immune cells and their subsets and related cytokines at the occurrence of UCBT PES. The changes in T, B, NK and mononuclear cells and its subgroups at the occurrence of PES; detection of patients before transplantation, On the day of transplantation, the number of white blood cells in peripheral blood at different time points, plasma C- reactive protein and serum related cytokines at different time points. Two, external stimulation experiments of different sources of transplantation: sST2 increased significantly at PES, and ST2 is an orphan receptor of IL-33, which is intended to promote PES effector cells from IL-33/ST2 signaling pathway. I L-33, sST2 stimulates fresh cord blood and peripheral blood stem cells (mobilized compatriots donor). Each type of cell is divided into 4 groups: experimental group: group A.IL-33+sST2; control group: B.sST2 group; C.IL-33 group; D. blank group. Cell count board is used to count the total number of cells before and after stimulation in vitro and after 72 hours in vitro. Flow cytometry is used to detect the cell subcell before and after stimulation. Results of group and ST2 receptor expression. Results the cell origin of PES after part one UCBT was studied by STR-PCR to detect the chimerism of umbilical cord blood and recipients before and after transplantation. The results showed that the chimerism of UCBT+7d donor in PES group was significantly higher than that of the non PES group, which was 70.58 + 22.86% and 37.18 + 18.96%. The two groups were among the two groups. Significant statistical differences, P0.0001. second part UCBT after the occurrence of PES effect cells, UCBT after PES, the detection of various immune cells and its subsets and related cytokines 1. leukocytes changes in the PES group and no PES group before the transplantation, the day of transplantation, the leukocyte count groups at the time of PES occurred, no statistical difference, P0.05.2.PES The cell subsets of T, B, NK and mononuclear cells were detected at the time of occurrence: CD45+CD3+T lymphocyte, CD45+CD3-CD19+B lymphocyte, CD45+CD3-CD56+NK cell, CD45+CD3-CD14+ mononuclear cells accounted for 90%, 0.3%, 2.1% and 0.2%.PES and 0.2%.PES without PES group T, B, NK and mononuclear cell subgroups There was no significant difference between the two groups in the PES group and the non PES group before the transplant, the day of the transplant, and the two groups without PES. The changes of serum cytokines in P0.05.4. were IL-6, MCP-1 and sST2 were significantly higher in the PES group than in the non PES group (P0.05). The cytokines IL-6 and MCP-1 of the patients with P0.05.PES were significantly decreased and recovered to the pre occurrence level (P0.01), while sST2 and PES were decreased, but there was no statistical difference (P0.05). There was no statistical difference between the two groups of IFN- gamma, IL-2, IL-1R alpha. (P0.05), at the same time, there was no significant difference in the expression level of the patients in group PES at different time points (P0.05). Two, the number of 1. cells in the external stimulation experiment of different sources: the number of fresh cord blood and peripheral blood stem cells by IL-33, sST2 after stimulation, the number of cells before and after stimulation was not significantly increased (P0.05), two kinds of cells. The cells with different sources had polymorphic changes after the stimulation of 12h. After that, the changes in the morphological changes were not significant.2.T, B, NK and monocyte subgroups. After the fresh cord blood was extracted by Ficoll, the proportion of mononuclear cells extracted from umbilical blood was T lymphocyte 30-50%, B lymphocyte 1-3%, NK cell 5-8%, The T lymphocyte 20-35%, B lymphocyte 2-5% and NK cells 3-9% in the monocyte 5-13%. peripheral blood stem cells. After the monocyte 18-32%. was stimulated to cultivate 12,24 and 72h, there was no significant difference in the proportion of T, B, and monocyte subsets in the four groups. With the prolongation of culture time, there were a small amount of cell apoptosis. There was no significant difference in the proportion of cell subgroups (P0.05).3.T, B, NK and the expression of ST2 receptor in monocyte: the expression of ST2 receptor in fresh umbilical blood and peripheral blood stem cells was similar, the expression of ST2 receptor on mononuclear cells was as high as 53-90%, followed by the B lymphocyte subgroup of about 30-60%, T lymphocyte and NK cell subgroup low, respectively. There was no significant change in the expression of ST2 receptor in each cell subgroup of the four groups (P0.05), and there was no significant change in the ST2 expression of each cell subgroup of the cells at different time points in the experimental group (P0.05). Conclusion 1.UCBT PES was a immunization reaction caused by a variety of cytokines when the donor began to be implanted, and its effector cells were derived from donors and donors. Early chimerism is a high risk factor for the occurrence of PES..2. serum monocyte related chemokine (MCP-1) increases significantly at the time of PES, while lymphocyte related specific cytokines (IL-2, IL-4, IL-8, IL-17A) have no significant changes in the occurrence of PES. The clinical manifestations of PES are mainly in the skin, intestinal and other tissues, and infer the single tissue. The nucleus macrophage may be the main effect cell of PES,.3. added to IL-33 and sST2 for simple culture in vitro, not the proliferation of immune cells and the increase of ST2 receptor expression, but it can not determine whether the level of cytokine changes. It is necessary to further detect the change of cytokine in the cell culture fluid and observe the IL-33/ST2 signaling pathway and sST2 Whether there is an effect on the signal transduction of the downstream cells to cause the secretion of cytokines, we also need to measure the activation of various immune cells, such as HLA-DR, CD27, CCR and so on, to provide the basis for revealing the mechanism of PES.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R457.7

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相關(guān)期刊論文 前5條

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本文編號(hào)：1783001