本文選題：NK細(xì)胞 + 急性移植物抗宿主病��； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2017年碩士論文

【摘要】：隨著醫(yī)療技術(shù)的飛速發(fā)展,異基因造血干細(xì)胞移植(allogeneic hematopoietic stem cell transplantation,allo-HSCT)后患者治愈率得到極大的提高。但由于缺乏有效預(yù)測指標(biāo)和治療措施,急性移植物抗宿主病(acute graft-versus-host disease,aGVHD),特別是激素耐藥的aGVHD,仍是制約allo-HSCT治愈率的關(guān)鍵技術(shù)性瓶頸[1]。aGVHD對靶細(xì)胞的損害可分為三步:(1)治療相關(guān)的損傷導(dǎo)致炎性細(xì)胞因子的大量釋放,從而啟動aGVHD;(2)T細(xì)胞激活后開始增殖活化;(3)T細(xì)胞攻擊患者上皮組織或器官。故aGVHD治療的關(guān)鍵在于抑制T細(xì)胞的增殖活化,但這也帶來了腫瘤復(fù)發(fā)和重癥感染的風(fēng)險[2,3]。自然殺傷(Natural killer,NK)細(xì)胞可快速殺傷患者體內(nèi)變異的細(xì)胞,具有抗腫瘤和抗感染的作用,對allo-HSCT具有重要意義。進(jìn)一步研究發(fā)現(xiàn),NK細(xì)胞在抗腫瘤、抗感染的同時,還具有抗aGVHD和促進(jìn)移植物植入的作用[4],為防治aGVHD帶來了新的希望。NK細(xì)胞在allo-HSCT后快速實現(xiàn)數(shù)量重建[5,6],此時NK細(xì)胞抗aGVHD作用是靠殺傷抗原呈遞細(xì)胞(antigen presenting cells,APCs),阻斷T細(xì)胞的活化而得以實現(xiàn)[7]。NK細(xì)胞同時表達(dá)抑制性和活化性受體,其對APCs的殺傷活性是由兩種受體與配體的結(jié)合情況共同決定的。當(dāng)APCs表面抑制性配體表達(dá)缺失,或活化性配體表達(dá)高于抑制性配體時,NK細(xì)胞將被激活,特異性的殺傷APCs。近年來的研究發(fā)現(xiàn),allo-HSCT后免疫重建的NK細(xì)胞表面的受體譜表達(dá)改變,特別是抑制性受體表達(dá)上調(diào),導(dǎo)致免疫重建的NK細(xì)胞功能受到抑制,殺傷靶細(xì)胞的活性低下。NK細(xì)胞功能抑制這一情況將持續(xù)到allo-HSCT后半年左右[8,9]。免疫重建的NK細(xì)胞作為一種功能被抑制的免疫細(xì)胞,是否還能殺傷APCs,是否還能有效發(fā)揮抗aGVHD作用,目前尚未明確。本研究通過分析NK細(xì)胞的重建情況,探討NK細(xì)胞重建與aGVHD的相關(guān)性,為進(jìn)一步研究重建NK細(xì)胞的抗aGVHD作用奠定了基礎(chǔ)。本研究首先觀察了患者allo-HSCT后NK細(xì)胞計數(shù)和活性與正常人的差異,分析了allo-HSCT后NK細(xì)胞計數(shù)和活性恢復(fù)情況,從而綜合評價NK細(xì)胞的重建。實驗思路簡述如下:選取2015年1月至2015年7月,在本院行allo-HSCT的26例患者,其中男性17例,女性9例,患者中位年齡37(12~62)歲。采集患者allo-HSCT后+30d、+60d、+90d時外周血作為實驗組,通過流式細(xì)胞儀(flowcytometry,FCM)檢測外周血中NK細(xì)胞計數(shù)和活性。將供者作為對照組,界定NK細(xì)胞計數(shù)和活性的正常值。將外周血單個核細(xì)胞(peripheral blood mononuclear cells,PBMCs)分離出外周血后計數(shù)。用CD45-APC、CD56-FITC、CD16-FITC和CD3-PerCP標(biāo)記PBMCs,FCM檢測出PBMNs中CD45、CD3、CD56、CD16的表達(dá)情況,計算CD3-CD56+CD16+的NK細(xì)胞計數(shù)。用CSFE熒光標(biāo)記K562細(xì)胞。將K562細(xì)胞單獨孵育作為對照組,觀察K562細(xì)胞自然凋亡率。將NK細(xì)胞與K562細(xì)胞按1:1比例共同培養(yǎng)作為實驗組,觀察NK細(xì)胞對K562細(xì)胞殺傷活性。對照組和實驗組細(xì)胞在培養(yǎng)箱中孵育2.5小時后用碘化丙啶(propidium Iodide,PI)標(biāo)記,FCM檢測CSFE+PI+的細(xì)胞(即凋亡的K562),最后計算出NK細(xì)胞殺傷活性。根據(jù)不同時間點NK細(xì)胞計數(shù)和活性的檢測結(jié)果,探索allo-HSCT后NK細(xì)胞數(shù)量和功能的重建情況。研究結(jié)果顯示:正常人NK細(xì)胞計數(shù)和活性為(1198±514)個/μl和(14.0±6.8)%�；颊遖llo-HSCT后+30d、+60d、+90d時NK細(xì)胞計數(shù)分別為(951±403)個/μl、(1358±418)個/μl和(1255±353)個/μl,與正常人無統(tǒng)計學(xué)差異�；颊逳K細(xì)胞活性分別為(7.3±3.7)%、(9.4±3.6)%和(9.3±2.4)%,較正常人顯著降低。以上結(jié)果說明,allo-HSCT后+30d時,NK細(xì)胞計數(shù)能恢復(fù)到正常人水平。但allo-HSCT后+90d內(nèi),NK細(xì)胞活性低下。NK細(xì)胞計數(shù)和活性在allo-HSCT后+30d時較+60d、+90d時低下。Allo-HSCT后NK細(xì)胞計數(shù)和活性的降低是否會影響其抗aGVHD作用,從而增加aGVHD發(fā)病的風(fēng)險則有待于進(jìn)一步研究。因此,本研究第二部分對26例患者進(jìn)行了跟蹤隨訪,以探索NK細(xì)胞重建對aGVHD發(fā)病的影響。實驗思路簡述如下:根據(jù)患者的臨床表現(xiàn)和相關(guān)病理指標(biāo),統(tǒng)計26例患者aGVHD發(fā)病、總體生存(overall survival,OS)和無進(jìn)展生存(progression-free survival,PFS)等臨床資料,分析NK細(xì)胞計數(shù)和活性與aGVHD發(fā)病和預(yù)后的相關(guān)性。研究結(jié)果顯示:根據(jù)患者是否發(fā)生aGVHD,將26例患者分為aGVHD組(11例)和無aGVHD組(15例)。與無aGVHD組相比,aGVHD組患者在allo-HSCT后+30d時NK細(xì)胞計數(shù)和活性顯著降低,即(655±216)個/μlvs(1169±372)個/μl(p=0.002)和(7.3±3.6)%vs(9.0±3.6)%(p=0.008)。根據(jù)患者是否發(fā)生Ⅱ~Ⅳ度aGVHD,進(jìn)一步將26例患者分為Ⅱ~Ⅳ度aGVHD組(7例)和0~Ⅰ度aGVHD組(19例)。與0~Ⅰ度aGVHD組相比,Ⅱ~Ⅳ度aGVHD組患者NK細(xì)胞計數(shù)和活性同樣顯著降低,且差異更為顯著,即(617±220)個/μl vs(1081±399)個/μl(p=0.001)和(4.2±1.7)%vs(8.3±3.5)%(p=0.001)。NK細(xì)胞計數(shù)(γ=—0.628,p=0.001)及活性(γ=—0.535,p=0.005)與aGVHD發(fā)病呈中度負(fù)相關(guān)。生存結(jié)果顯示,Ⅱ~Ⅳ度aGVHD組復(fù)發(fā)率顯著升高(57%vs 5%,p=0.010),且1年P(guān)FS率明顯下降(43%vs 84%,p=0.010)。Ⅱ~Ⅳ度aGVHD組患者的1年OS率同樣低于0~Ⅰ度aGVHD組(71%vs 90%),但無統(tǒng)計學(xué)差異(p=0.374)。以上結(jié)果說明,allo-HSCT后發(fā)生aGVHD的患者+30d時NK細(xì)胞計數(shù)和活性較未發(fā)生aGVHD的患者顯著降低,且發(fā)生Ⅱ~Ⅳ度aGVHD患者的NK細(xì)胞計數(shù)和活性降低更為顯著。Allo-HSCT后+30d時NK細(xì)胞計數(shù)和活性與aGVHD呈中度負(fù)相關(guān)性。+30d時NK細(xì)胞計數(shù)和活性低下的aGVHD患者,其復(fù)發(fā)率顯著高,PFS率顯著降低。綜上,本研究結(jié)果顯示,allo-HSCT后+30d時,NK細(xì)胞完成數(shù)量重建,但+90d內(nèi)NK細(xì)胞活性顯著低于正常人水平。Allo-HSCT后+30d時,重建NK細(xì)胞的計數(shù)和活性處于最低水平,且與aGVHD的發(fā)生和預(yù)后存在一定聯(lián)系,通過早期監(jiān)測NK細(xì)胞計數(shù)和活性可識別aGVHD高�；颊�,這將為防治aGVHD提供新的思路。
[Abstract]:With the rapid development of medical technology, the cure rate of allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been greatly improved. However, due to the lack of effective predictors and treatment measures, acute graft-versus-host disease (acute graft-versus-host disease, aGVHD), especially hormone Drug-resistant aGVHD is still a key technical bottleneck that restricts the cure rate of allo-HSCT, [1].aGVHD can be divided into three steps: (1) the treatment related injury leads to the massive release of inflammatory cytokines, thus initiating aGVHD; (2) T cells begin to proliferate and activate; (3) T cells attack the epithelial tissue or organs of the patients. So aGVHD treatment The key is to inhibit the proliferation and activation of T cells, but this also brings the risk of tumor recurrence and severe infection. The [2,3]. natural killer (Natural killer, NK) cells can quickly kill the mutant cells in the patient's body. It has the role of anti-tumor and anti infection, and is of great significance to allo-HSCT. Further studies have found that NK cells are anti tumor and anti infection. At the same time, it also has the effect of resisting aGVHD and promoting graft implantation, which brings new hope that.NK cells can quickly reconstruct [5,6] after allo-HSCT for the prevention and control of aGVHD. At this time, the anti aGVHD effect of NK cells is by killing antigen presenting cells (antigen presenting cells, APCs) and blocking the activation of.NK cells. Expression of inhibitory and activated receptors, whose killing activity against APCs is determined by the combination of two receptors and ligands. When the expression of APCs surface inhibitive ligand is missing, or the expression of active ligand is higher than the inhibitory ligand, the NK cells will be activated. The specific killing of APCs. in recent years has found that the immune weight after allo-HSCT is heavy. The changes in the expression of the receptor spectrum on the surface of the NK cells, especially the up regulation of the inhibitory receptor expression, resulted in the inhibition of the function of the immune rebuilt NK cells and the inhibition of the function inhibition of the low active.NK cells of the target cells. This situation will continue to the NK cells reconstructed by [8,9]. immunized as a function that is suppressed in the second half of allo-HSCT. Cell, whether it can kill APCs, and whether it can play an effective anti aGVHD effect, is not clear. This study is to explore the correlation between NK cell reconstruction and aGVHD by analyzing the reconstruction of NK cells. This study has laid the foundation for further study of the anti aGVHD effect of reconstruction of NK cells. The difference between sex and normal people, analysis of the NK cell count and activity recovery after allo-HSCT, and thus comprehensive evaluation of the reconstruction of NK cells. The experimental ideas are summarized as follows: 26 cases of allo-HSCT in our hospital from January 2015 to July 2015 were selected, including 17 men, 9 women and 37 (12~62) years of middle age. After allo-HSCT +30d The peripheral blood was used as the experimental group at +60d and +90d as the experimental group. The count and activity of NK cells in peripheral blood were detected by flow cytometry (flowcytometry, FCM). The donors were used as the control group to define the normal values of NK cell count and activity. The peripheral blood mononuclear cells (peripheral blood mononuclear cells, PBMCs) were separated out of the peripheral blood count. 6-FITC, CD16-FITC and CD3-PerCP were labeled PBMCs, FCM was used to detect the expression of CD45, CD3, CD56, CD16 in PBMNs, and the CD3-CD56+CD16+ NK cell count was calculated. The cytotoxic activity of NK cells to K562 cells was observed. The cells in the control group and the experimental group were incubated in the incubator for 2.5 hours and were labeled with propidium Iodide (PI), and FCM was used to detect the CSFE+PI+ cells (the apoptotic K562). Finally, the NK cell killing activity was calculated. According to the detection results of the count and activity of the non simultaneous NK cells, the allo-HSCT was explored. The results of the number and function of the post NK cells showed that the count and activity of NK cells in normal people were (1198 + 514) / mu L and (14 + 6.8)%. The count of NK cells was (951 + 403) / u l, (1358 + 418) / L and (1255 + 353) / mu l at +30d, +60d and +90d after allo-HSCT, and there was no statistical difference from normal people. (7.3 + 3.7)%, (9.4 + 3.6)% and (9.3 + 2.4)%, significantly lower than normal people. The results showed that the number of NK cells could be restored to normal level at +30d after allo-HSCT, but in +90d after allo-HSCT, the count and activity of.NK cells in NK cells were lower than +60d after allo-HSCT, and the count and activity of the cells after.Allo-HSCT were low. The second part of this study was followed up to explore the effect of NK cell reconstruction on the incidence of aGVHD. The second part of this study was followed up to explore the effect of NK cell reconstruction on the incidence of aGVHD. The experimental ideas were summarized as follows: according to the patient's clinical manifestation and the related pathological indexes, 26 cases were counted. Patients with aGVHD, overall survival (overall survival, OS) and progression free survival (progression-free survival, PFS) were used to analyze the correlation between the count and activity of NK cells and the morbidity and prognosis of aGVHD. The results showed that 26 patients were divided into aGVHD group (11 cases) and no aGVHD group (15 cases) according to the occurrence of aGVHD. Compared with group aGVHD, the count and activity of NK cells decreased significantly at +30d after allo-HSCT, namely (655 + 216) / mu LVS (1169 + 372) / mu L (p=0.002) and (7.3 + 3.6)%vs (9 + 3.6)% (p=0.008). According to the occurrence of patients with II to IV degree aGVHD, 26 patients were further divided into class II ~ IV aGVHD group (7 cases) and 0~ I degree group (19 cases). Compared with group aGVHD, the count and activity of NK cells in group II ~ IV degree aGVHD were also significantly decreased, and the difference was more significant, that is, (617 + 220) / mu l vs (1081 + 399) / mu L (p=0.001) and (4.2 + 1.7)%vs (8.3 + 3.5)% (p=0.001).NK cell count (gamma = 0.628, p= 0.001) and activity (gamma = 0.535, p=0.005) had a moderate negative correlation with the onset of disease. The results showed that the recurrence rate of group II ~ IV aGVHD group was significantly higher (57%vs 5%, p=0.010), and the 1 year PFS rate decreased significantly (43%vs 84%, p=0.010). The 1 year OS rate in group II ~ IV aGVHD group was also lower than 0~ I aGVHD group (71%vs 90%), but there was no statistical difference (p=0.374). The patients who had no aGVHD significantly decreased, and the number and activity of NK cells in patients with 2 ~ IV degree aGVHD were more significant than those of NK cell count and NK cell count and activity with aGVHD, and the NK cell count and low activity of aGVHD were significantly higher and the PFS rate decreased significantly. The results showed that the number of NK cells was rebuilt at +30d after allo-HSCT, but when the activity of NK cells in +90d was significantly lower than that of normal human.Allo-HSCT, the count and activity of NK cells were at the lowest level, and there was a relationship with the occurrence and prognosis of aGVHD, and the aGVHD high-risk patients could be identified by early monitoring of NK cell count and activity. This will provide a new way of thinking for the prevention and control of aGVHD.

【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】：碩士
【學(xué)位授予年份】：2017
【分類號】：R457.7

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