本文關鍵詞：NK細胞受體與宿主MHCⅠ類分子匹配程度對骨髓移植后造血重建的影響　出處：《中國人民解放軍軍事醫(yī)學科學院》2015年碩士論文　論文類型：學位論文

  更多相關文章： 異基因骨髓移植 NK細胞 重建造血功能 移植物抗宿主病 組織相容復合物

【摘要】：自然殺傷細胞(natural killer cell, NK細胞)是機體天然免疫細胞,可以清除體內(nèi)變異細胞,包括惡性病變和被病原體感染的細胞,可直接識別機體內(nèi)的正常和異常細胞,進而對機體內(nèi)的異常信號做出快速反應。一般認為,NK細胞通過其細胞表面的NK細胞受體來區(qū)分正常和異常細胞。NK細胞表面表達NK細胞免疫球蛋白受體(注釋：killer cell immunoglobulin-like receptor, KIR),能特異性識別并結合其他細胞表面的主要組織相容性復合體(注釋：1najor histocompatibility complex, MHC)I類分子。KIR分為抑制型和活化型兩種,分別向細胞內(nèi)傳遞抑制性信號和活化性信號,共同調(diào)節(jié)NK細胞的殺傷活性。目前公認的理論為‘'Missing Self"機制：機體的正常細胞表達大量的MHCI類分子,他們與NK細胞表面的抑制型KIR結合,抑制NK細胞活性；在某些癌變或者病毒感染細胞MHC I類分子表達減少時,不能有效抑制NK細胞的殺傷功能,導致其被活化的NK細胞清除。在臨床中骨髓移植(注釋：b one marrow transplantation, B M T)是治療血液系統(tǒng)中良惡性疾病的有效手段。有研究得出來源于供者的NK細胞可以清除受者體內(nèi)的白血病細胞。本課題組前期研究表明,NK細胞可以減少移植物抗宿主病的發(fā)生,同時因為NK細胞的生物學效應往往依賴于其受體與配體的識別及匹配程度,故該效應與自然殺傷細胞KIR和受者MHC I類分子匹配程度有關。本研究重點關注NK細胞KIR與MHCI類分子的匹配程度對骨髓移植后造血重建的影響研究。NK細胞作為稀有淋巴細胞亞群,在外周血和脾臟中含量極少,想要獲得足夠治療用量的NK細胞,需要對其進行分離純化和體外擴增培養(yǎng)。本研究中我們使用密度梯度離心和免疫磁珠(MACS)兩種NK細胞分選技術,和使用血小板裂解液(platelet lysate, PL)對NK細胞進行體外擴增培養(yǎng)的方法,獲得了大量高純度的NK細胞。為了進一步研究allo-NK細胞在骨髓移植中的作用,我們擬采用動物模型來觀察allo-NK細胞受體與宿主MHCI類分子的匹配程度對骨髓移植后移植物植入的影響和機制。進行該研究首先要建立骨髓移植模型,移植前的預處理是供體植入的必要條件,不同的預處理強度對骨髓移植預后具有顯著影響。本研究中我們選用全身輻照(注釋：total body irradiation, TBI)進行預處理,利用3Gy和6Gy不同強度60Coγ射線全身照射BALB/C和CB6F1雌性小鼠,制備成清髓和非清髓兩種模型,然后通過尾靜脈輸入供者C57BL/6J雄性小鼠的骨髓細胞,通過觀察受者的生存期、嵌合率等情況,確定骨髓移植動物模型的最佳預處理強度。研究結果6Gy預處理的對照組小鼠在觀察期內(nèi)全部死亡,進行骨髓移植后的實驗組小鼠生存率在90%以上,并且6Gy預處理的骨髓移植小鼠外周血中的白細胞和血小板水平適宜進行造血重建的觀察。為了觀察供鼠NK細胞受體與受鼠MHCI類分子匹配程度對骨髓移植小鼠造血重建的影響。我們分離了30只C57BL/c小鼠的骨髓細胞和NK細胞,輸給經(jīng)過預處理的BALB/c和CB6F1小鼠,受鼠被分為單純輻照組、輸入骨髓細胞組和輸入骨髓細胞及NK細胞組三組,6只／組,觀察不同時間點血常規(guī)、生存時間、病理組織學等指標變化,進行組間比較。結果顯示：輸入骨髓細胞、骨髓細胞及NK細胞組小鼠均長期存活,也未見GVHD病理學改變。移植后第14天時,輸入骨髓細胞組和輸入骨髓細胞及NK細胞組的BALB/c小鼠(MHC工類分子完全不相合)外周血白細胞計數(shù)分別為：(1.35±0.33)×109/L和(1.80±0.18)×109/L,兩組之間具有顯著性差異,血小板計數(shù)分別為：(79±19)×109/L和(117±13)×109/L,兩組之間具有顯著性差異；輸入骨髓細胞組和輸入骨髓細胞及NK細胞組的CB6F1小鼠(MHC I類分子半相合)外周血白細胞計數(shù)分別為：(1.52±0.26)×109/L和(1.85±0.34)×109/L,兩組之間差異無統(tǒng)計學意義,血小板計數(shù)分別為(90±12)×109/L和(113±15)×109/L,兩組之間差異具有統(tǒng)計學意義；BALB/c小鼠白細胞的改善略優(yōu)于CB6F1小鼠。由此得出結論allo-NK細胞可以促進供受者移植術后恢復功能造血,且其程度受NK細胞表面受體和MHC 1類分子匹配程度的影響,而對造血功能的影響只體現(xiàn)在移植后造血重建的早期,這也可能與輸入的NK細胞半衰期有關。
[Abstract]:Natural killer cells (natural killer cell, NK cells) is the body's natural immune cells, can remove the variation in body cells, including malignant lesions and cells infected by pathogens, can directly identify the normal and abnormal cells in the organism, and the abnormal signal for the body's rapid response. It is generally considered that NK cells through its cell surface the NK cell receptor to distinguish between normal and abnormal.NK cell surface expression of NK cell immunoglobulin receptors (Note: killer cell immunoglobulin-like receptor, KIR), which could specifically recognize and bind the organization he cell surface MHC (Note: 1najor histocompatibility complex, MHC) I molecules.KIR inhibitory type and the activation of two kinds, respectively transmit inhibitory signals and activating signal into cells, regulate the cytotoxic activity of NK cells. The currently accepted theory "'Missing Self" mechanism: normal body cells expression of MHCI molecules, they combined with the inhibitory KIR cell surface NK, inhibit the activity of NK cells; expression in some cancer or virus infected cells MHC class I molecules is reduced, can not effectively inhibit NK cell killing function, result in the activation of NK bone marrow cell clearance in clinical transplantation. (Note: B one marrow transplantation, B M T) is an effective means in the treatment of blood system diseases. Research comes from donor NK cells can be removed by white blood cells in vivo disease. Ourprevious studies showed that NK cells can to reduce the incidence of graft-versus-host disease, at the same time as the recognition of biological effects of NK cells often depends on its receptors and ligands and the matching degree, the effect of natural killer cell KIR and recipient MHC class I molecule matching degree Study on the effect of.NK cells. This study focuses on the matching degree of NK cell KIR and MHCI molecules on the hematopoietic reconstitution after bone marrow transplantation as rare lymphocyte subsets, rarely content in peripheral blood and spleen, want to get enough treatment dosage of NK cells, the need for purification and amplification in vitro. In this study, we use density gradient centrifugation and immunomagnetic beads (MACS) two NK cell sorting technique, and the use of platelet lysate (platelet lysate, PL) on NK cells in vitro, to obtain a large number of high purity of NK cells. In order to further study the allo-NK cells in bone marrow transplantation. We intend to use the animal model to observe the mechanism and the matching degree of allo-NK cell receptor and host MHCI molecules on bone marrow transplantation after graft implantation. The first study to establish bone marrow Transplantation model, pretreatment before transplantation is a necessary condition for donor implantation, pretreatment of different intensity has a significant impact on the prognosis of bone marrow transplantation. In this study we used whole-body irradiation (Note: total body irradiation, TBI) were pretreated by 3Gy and 6Gy with different intensity of 60Co gamma ray irradiation BALB/C and female CB6F1 mice were prepared into myeloablative and non myeloablative two models, and then through the tail vein input donor C57BL/6J male mice bone marrow cells, by observing the recipient survival and chimerism etc., to determine the optimum pretreatment intensity of bone marrow transplantation animal model. The mice of control group results 6Gy pretreatment all died in the observation period, after bone marrow transplantation in the experimental group, the survival rate is more than 90%, in mice after bone marrow transplantation and 6Gy pretreatment of peripheral white blood cells and platelet levels suitable for hematopoietic reconstitution Observation. In order to observe the donor NK cell receptor and rat MHCI molecules affected by the matching degree of hematopoietic reconstitution in mice after bone marrow transplantation. We isolated bone marrow cells and NK cells in 30 C57BL/c mice, lost after the pretreatment of BALB/c and CB6F1 mice, rats were divided into simple irradiation group, bone marrow cell input group and input bone marrow cells and NK cells in three groups, 6 rats in each group were observed at different time points, blood routine, survival time, histopathological changes were compared between groups. Results showed that the input of bone marrow cells, bone marrow cells and NK cells of mice survived for a long time, there were no GVHD pathology. Fourteenth days of transplantation when the input and input of bone marrow cells in bone marrow cell group and NK cell group BALB/c mice (MHC molecules completely mismatched) peripheral white blood cell counts were: (1.35 + 0.33) * 109/L and (1.80 + 0.18) * 109/L, between the two groups Significant differences in platelet count, respectively: (79 + 19) * 109/L and (117 + 13) * 109/L, with a significant difference between the two groups; the input and input of bone marrow cells bone marrow cell group and NK cell group CB6F1 mice (MHC class I molecules haploidentical peripheral white blood cell count) respectively. For: (1.52 + 0.26) * 109/L and (1.85 + 0.34) * 109/L, no significant difference between the two groups, platelet counts were (90 + 12) * 109/L and (113 + 15) * 109/L, the difference was statistically significant between the two groups; BALB/c mice improved slightly better than that of CB6F1 mice. Conclusion allo-NK cells can promote the recovery of hematopoietic function for recipients, and the degree of influence by NK cell surface receptors and MHC 1 molecules, the degree of influence on hematopoietic function only in hematopoietic reconstitution after transplantation in the early stage, it can also enter the NK cells and the half decay It is related to the period.

【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R457.7

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