NK细胞受体与宿主MHCⅠ类分子匹配程度对骨髓移植后造血重建的影响

发布时间：2018-01-05 02:00

本文关键词：NK细胞受体与宿主MHCⅠ类分子匹配程度对骨髓移植后造血重建的影响　出处：《中国人民解放军军事医学科学院》2015年硕士论文　论文类型：学位论文

【摘要】：自然杀伤细胞(natural killer cell, NK细胞)是机体天然免疫细胞,可以清除体内变异细胞,包括恶性病变和被病原体感染的细胞,可直接识别机体内的正常和异常细胞,进而对机体内的异常信号做出快速反应。一般认为,NK细胞通过其细胞表面的NK细胞受体来区分正常和异常细胞。NK细胞表面表达NK细胞免疫球蛋白受体(注释：killer cell immunoglobulin-like receptor, KIR),能特异性识别并结合其他细胞表面的主要组织相容性复合体(注释：1najor histocompatibility complex, MHC)I类分子。KIR分为抑制型和活化型两种,分别向细胞内传递抑制性信号和活化性信号,共同调节NK细胞的杀伤活性。目前公认的理论为‘'Missing Self"机制：机体的正常细胞表达大量的MHCI类分子,他们与NK细胞表面的抑制型KIR结合,抑制NK细胞活性；在某些癌变或者病毒感染细胞MHC I类分子表达减少时,不能有效抑制NK细胞的杀伤功能,导致其被活化的NK细胞清除。在临床中骨髓移植(注释：b one marrow transplantation, B M T)是治疗血液系统中良恶性疾病的有效手段。有研究得出来源于供者的NK细胞可以清除受者体内的白血病细胞。本课题组前期研究表明,NK细胞可以减少移植物抗宿主病的发生,同时因为NK细胞的生物学效应往往依赖于其受体与配体的识别及匹配程度,故该效应与自然杀伤细胞KIR和受者MHC I类分子匹配程度有关。本研究重点关注NK细胞KIR与MHCI类分子的匹配程度对骨髓移植后造血重建的影响研究。NK细胞作为稀有淋巴细胞亚群,在外周血和脾脏中含量极少,想要获得足够治疗用量的NK细胞,需要对其进行分离纯化和体外扩增培养。本研究中我们使用密度梯度离心和免疫磁珠(MACS)两种NK细胞分选技术,和使用血小板裂解液(platelet lysate, PL)对NK细胞进行体外扩增培养的方法,获得了大量高纯度的NK细胞。为了进一步研究allo-NK细胞在骨髓移植中的作用,我们拟采用动物模型来观察allo-NK细胞受体与宿主MHCI类分子的匹配程度对骨髓移植后移植物植入的影响和机制。进行该研究首先要建立骨髓移植模型,移植前的预处理是供体植入的必要条件,不同的预处理强度对骨髓移植预后具有显著影响。本研究中我们选用全身辐照(注释：total body irradiation, TBI)进行预处理,利用3Gy和6Gy不同强度60Coγ射线全身照射BALB/C和CB6F1雌性小鼠,制备成清髓和非清髓两种模型,然后通过尾静脉输入供者C57BL/6J雄性小鼠的骨髓细胞,通过观察受者的生存期、嵌合率等情况,确定骨髓移植动物模型的最佳预处理强度。研究结果6Gy预处理的对照组小鼠在观察期内全部死亡,进行骨髓移植后的实验组小鼠生存率在90%以上,并且6Gy预处理的骨髓移植小鼠外周血中的白细胞和血小板水平适宜进行造血重建的观察。为了观察供鼠NK细胞受体与受鼠MHCI类分子匹配程度对骨髓移植小鼠造血重建的影响。我们分离了30只C57BL/c小鼠的骨髓细胞和NK细胞,输给经过预处理的BALB/c和CB6F1小鼠,受鼠被分为单纯辐照组、输入骨髓细胞组和输入骨髓细胞及NK细胞组三组,6只／组,观察不同时间点血常规、生存时间、病理组织学等指标变化,进行组间比较。结果显示：输入骨髓细胞、骨髓细胞及NK细胞组小鼠均长期存活,也未见GVHD病理学改变。移植后第14天时,输入骨髓细胞组和输入骨髓细胞及NK细胞组的BALB/c小鼠(MHC工类分子完全不相合)外周血白细胞计数分别为：(1.35±0.33)×109/L和(1.80±0.18)×109/L,两组之间具有显著性差异,血小板计数分别为：(79±19)×109/L和(117±13)×109/L,两组之间具有显著性差异；输入骨髓细胞组和输入骨髓细胞及NK细胞组的CB6F1小鼠(MHC I类分子半相合)外周血白细胞计数分别为：(1.52±0.26)×109/L和(1.85±0.34)×109/L,两组之间差异无统计学意义,血小板计数分别为(90±12)×109/L和(113±15)×109/L,两组之间差异具有统计学意义；BALB/c小鼠白细胞的改善略优于CB6F1小鼠。由此得出结论allo-NK细胞可以促进供受者移植术后恢复功能造血,且其程度受NK细胞表面受体和MHC 1类分子匹配程度的影响,而对造血功能的影响只体现在移植后造血重建的早期,这也可能与输入的NK细胞半衰期有关。
[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.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R457.7

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