外周血造血干细胞来源微泡的生物学特性

发布时间：2018-01-10 10:34

本文关键词：外周血造血干细胞来源微泡的生物学特性　出处：《中国人民解放军军事医学科学院》2017年硕士论文　论文类型：学位论文

【摘要】：造血干细胞(hematopoietic stem cells)是一类异质性的、具有自我更新和多向血细胞分化潜能的成体干细胞。根据来源不同,其可分类为骨髓造血干细胞,脐血造血干细胞,胎肝造血干细胞和外周血造血干细胞。因外周血造血干细胞(Peripheral blood hematopoietic stem cells,PB-HSC)具有采集方法简便,短期内可重复采集,受者输注后造血、免疫功能重建快,且植入率高等优点,在移植和细胞免疫治疗方面发挥日益重要的作用。微泡(microvesicles,MV)是细胞分泌的亚微米双层膜结构,包括从内涵体分泌的外泌体,以及从绝大多数细胞膜表面分泌的微粒。其含有母体细胞的蛋白质、脂质、mRNA以及microRNA等成分,通过与其它细胞融合,将其内容物释放到靶细胞,影响其功能,从而在不同细胞间信息传递过程中发挥重要作用。微泡可由多种细胞分泌,并广泛分布于血浆、唾液、乳汁、汗液等各种体液中。不仅正常细胞可分泌微泡,肿瘤细胞也可以分泌微泡:在肿瘤病人的体液中可以检测到微泡。因此在生理和病理状态下,不同细胞分泌的微泡是一个混合的群体。微泡随着细胞的来源、数量、大小和抗原成分的变化而不同。外周血造血干细胞具有免疫调节,促进造血恢复等作用。因为微泡带有母体细胞的信息,所以我们推断外周干来源的微泡可能也具有同样的功能。而且研究表明,微泡没有或者具有很小的免疫原性,如果其具有一定的功能,可具有广泛的临床应用。但是,目前对外周血造血干细胞来源的微泡研究甚少。本研究旨在对外周血造血干细胞来源的微泡进行生物学特性探究,尤其对其免疫调节和促造血功能加以探索。研究目的提取并鉴定正常人外周血造血干细胞来源的微泡,并研究其生物学特性:包括探究正常人外周血造血干细胞来源的微泡的免疫调节功能及对造血集落形成的影响。研究内容将正常人外周血造血干细胞提取并培养,从培养上清中提取出外周血造血干细胞来源的微泡并鉴定,研究其生物学特性:探究正常人外周血造血干细胞来源的微泡对外周血单个核细胞是否具有免疫调节功能?及正常人外周血造血干细胞来源的微泡是否对造血集落形成有影响?研究方法(1)利用密度梯度离心法分离正常人动员后外周血造血干细胞,收集培养第48小时上清液,超速离心法分离提取微泡;(2)通过电子显微镜观察微泡形态;采用bicinchoninic acid(BCA)法标定微泡数量;采用流式细胞仪检测其表面标志物;(3)将微泡与正常人外周血单个核细胞(peripheral blood mononuclear cells,PB-MNC)共培养12h后,通过共聚焦扫描电镜观察微泡与单个核细胞的作用方式;共培养48h后采用酶联免疫吸附试验法(ELISA)法检测上清中IL-2,IL-6,IL-8,IL-10,IFN-γ及TNF-α的分泌量;共培养48h后采用流式细胞仪检测T细胞亚群及T细胞激活变化;共培养48h后采用流式细胞仪检测不同亚群细胞胞内细胞因子染色情况;(4)采用甲基纤维素半固体培养基检测微泡及微泡与单个核细胞共培养48小时后上清液对外周血造血干细胞集落形成的影响。研究结果通过密度梯度离心法可提取出动员后外周血造血干细胞。利用超速离心法可成功提取出外周血造血干细胞来源的微泡。透射电子显微镜观察分离得到的微泡为卵圆形膜性小囊泡。流式细胞术测得微泡高表达其特异性标志CD63(85.86%),并带有其母体细胞标志如干细胞标志CD34(33.52%),T细胞标志CD3(43.98%)、CD45RA(23.54%)、CD45RO(68.3%),NK细胞标志CD56(32.32%)等。共聚焦扫描电镜显示微泡可以与外周血单个核细胞相融合并促进其分泌IL-6,IL-8,IL-10与TNF-α细胞因子。流式细胞仪检测结果显示T细胞亚群及T细胞激活无明显改变。胞内因子染色结果表明CD4+,CD8+,CD11c+细胞内因子无明显改变。集落培养实验表明微泡及微泡与单个核细胞共培养48小时后上清液很可能具有促进造血集落形成的作用。研究结论外周血造血干细胞来源的微泡具有免疫调节及可能促进造血集落形成的作用。
[Abstract]:Hematopoietic stem cells (hematopoietic stem cells) is a kind of heterogeneity, self-renewal and multilineage differentiation potential of blood cells of adult stem cells. According to different sources, which can be classified as bone marrow hematopoietic stem cells, hematopoietic stem cells, fetal liver hematopoietic stem cells and peripheral blood stem cells for peripheral. Hematopoietic stem cells (Peripheral blood hematopoietic stem cells, PB-HSC) with sampling method is simple, the short term can be collected, recipients after infusion of hematopoiesis, immune reconstitution, and implantation rate is high, play an increasingly important role in cell transplantation and immunotherapy. Microbubbles (microvesicles, MV) is sub micron double membrane cells, including exosomes secreted from endosomes, and from the vast majority of particle surface membrane secretion. It contains maternal cell protein, lipid, mRNA and microRNA and other ingredients, through Fusion with other cells, will release their content into the target cells, affect its function, resulting in different communication between cells plays an important role in the process. The micro bubble secreted by many cells, and is widely distributed in the plasma, saliva, milk, sweat and other various body fluids. Not only the normal cells can secrete microbubbles. Tumor cells can also secrete microbubbles in tumor patients in body fluids can detect microbubbles. Therefore in physiological and pathological conditions, different cell secretion of microbubbles is a mixture of groups. Microbubbles with the cell source, quantity, size and changes in antigenic components and different. Peripheral blood stem cells with immune regulation, promote the recovery of hematopoietic function. Because microbubbles with maternal cell information, so we infer that peripheral stem derived microvesicles may have the same function. And the research shows that the micro bubble has little or no free Immunogenicity, if it has a certain function, may have a wide clinical application. However, the peripheral blood hematopoietic stem cells derived microvesicles little studied. This study aimed to peripheral blood hematopoietic stem cells derived microvesicles on biological characteristics of research, especially on the immune regulation and hematopoietic function to explore. The purpose of the study. Microbubble extraction and identification of normal human peripheral blood hematopoietic stem cells, and to study its biological characteristics, including immune microbubbles on human peripheral blood hematopoietic stem cell source regulating function and colony forming effect on hematopoiesis. Research content of normal peripheral blood hematopoietic stem cells isolation and culture, culture and extraction from peripheral blood hematopoietic stem cells derived microvesicles and identified in the supernatant, and study its biological characteristics: the study of normal human peripheral blood hematopoietic stem cells derived microvesicles in peripheral blood mononuclear cells are Whether they can regulate immune function? And normal peripheral blood hematopoietic stem cells derived microvesicles have effects on hematopoietic colony formation is set? Research methods (1) using density gradient centrifugation of normal mobilized peripheral blood hematopoietic stem cells cultured for forty-eighth hours, collect the supernatant, ultra centrifugation separation and extraction of micro bubble; (2) using electron microscope to observe the micro bubble morphology; using bicinchoninic acid (BCA) calibration method was used to detect the number of microbubbles; its surface markers by flow cytometry; (3) the microbubble blood mononuclear cells and normal human peripheral (peripheral blood mononuclear cells, PB-MNC) after 12h co cultured by confocal scanning electron microscope observation of microbubbles and mononuclear cells; after 48h co cultured by enzyme linked immunosorbent assay (ELISA) method for the detection of IL-2, IL-6 in the supernatant, IL-8, IL-10, IFN- and TNF- secretion quantity of gamma alpha; after 48h co cultured by flow cytometry Detection of T cell subsets and T cell activation changes; co cultured 48h were detected by flow cytometry in different Ya Qun cell intracellular cytokine staining; (4) using methylcellulose semisolid medium to detect microbubbles and effect of microbubble supernatant after 48 hours of peripheral blood hematopoietic stem cell colony formation of co cultured with mononuclear cells. Results by density gradient centrifugation can be extracted after the mobilization of peripheral blood stem cells can be successfully extracted from microbubbles. Peripheral blood hematopoietic stem cells derived by ultracentrifugation. Isolated microvesicles oval membranous vesicles were observed by transmission electron microscope. Flow cytometry test micro global high expression of the specific markers of CD63 (85.86%), and with its parent cell markers such as stem cell marker CD34 (33.52%), T (43.98%), CD3 cell marker CD45RA (23.54%), CD45RO (68.3%), NK (32.32%) CD56 cell markers. Confocal scanning electron microscopy showed that the microbubble in peripheral blood mononuclear cells and promote the secretion of IL-6 blending with IL-8, IL-10, TNF- and alpha cytokines. Flow cytometry showed that T cell subsets and activation of T cells had no obvious change. Intracellular cytokine staining results showed that CD4+, CD8+, CD11c+ cell factor no obvious change. Colony culture experiments show that microbubbles and microbubbles and mononuclear cells were cultured for 48 hours after a supernatant could promote the hematopoietic colony formation. The conclusion of the study of peripheral blood hematopoietic stem cells derived microvesicles have immunomodulatory and may promote hematopoietic colony formation.

【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】：2017
【分类号】：R457.7

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