本文關(guān)鍵詞：外周血造血干細(xì)胞來源微泡的生物學(xué)特性　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2017年碩士論文　論文類型：學(xué)位論文

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【摘要】：造血干細(xì)胞(hematopoietic stem cells)是一類異質(zhì)性的、具有自我更新和多向血細(xì)胞分化潛能的成體干細(xì)胞。根據(jù)來源不同,其可分類為骨髓造血干細(xì)胞,臍血造血干細(xì)胞,胎肝造血干細(xì)胞和外周血造血干細(xì)胞。因外周血造血干細(xì)胞(Peripheral blood hematopoietic stem cells,PB-HSC)具有采集方法簡便,短期內(nèi)可重復(fù)采集,受者輸注后造血、免疫功能重建快,且植入率高等優(yōu)點,在移植和細(xì)胞免疫治療方面發(fā)揮日益重要的作用。微泡(microvesicles,MV)是細(xì)胞分泌的亞微米雙層膜結(jié)構(gòu),包括從內(nèi)涵體分泌的外泌體,以及從絕大多數(shù)細(xì)胞膜表面分泌的微粒。其含有母體細(xì)胞的蛋白質(zhì)、脂質(zhì)、mRNA以及microRNA等成分,通過與其它細(xì)胞融合,將其內(nèi)容物釋放到靶細(xì)胞,影響其功能,從而在不同細(xì)胞間信息傳遞過程中發(fā)揮重要作用。微泡可由多種細(xì)胞分泌,并廣泛分布于血漿、唾液、乳汁、汗液等各種體液中。不僅正常細(xì)胞可分泌微泡,腫瘤細(xì)胞也可以分泌微泡:在腫瘤病人的體液中可以檢測到微泡。因此在生理和病理狀態(tài)下,不同細(xì)胞分泌的微泡是一個混合的群體。微泡隨著細(xì)胞的來源、數(shù)量、大小和抗原成分的變化而不同。外周血造血干細(xì)胞具有免疫調(diào)節(jié),促進(jìn)造血恢復(fù)等作用。因為微泡帶有母體細(xì)胞的信息,所以我們推斷外周干來源的微泡可能也具有同樣的功能。而且研究表明,微泡沒有或者具有很小的免疫原性,如果其具有一定的功能,可具有廣泛的臨床應(yīng)用。但是,目前對外周血造血干細(xì)胞來源的微泡研究甚少。本研究旨在對外周血造血干細(xì)胞來源的微泡進(jìn)行生物學(xué)特性探究,尤其對其免疫調(diào)節(jié)和促造血功能加以探索。研究目的提取并鑒定正常人外周血造血干細(xì)胞來源的微泡,并研究其生物學(xué)特性:包括探究正常人外周血造血干細(xì)胞來源的微泡的免疫調(diào)節(jié)功能及對造血集落形成的影響。研究內(nèi)容將正常人外周血造血干細(xì)胞提取并培養(yǎng),從培養(yǎng)上清中提取出外周血造血干細(xì)胞來源的微泡并鑒定,研究其生物學(xué)特性:探究正常人外周血造血干細(xì)胞來源的微泡對外周血單個核細(xì)胞是否具有免疫調(diào)節(jié)功能?及正常人外周血造血干細(xì)胞來源的微泡是否對造血集落形成有影響?研究方法(1)利用密度梯度離心法分離正常人動員后外周血造血干細(xì)胞,收集培養(yǎng)第48小時上清液,超速離心法分離提取微泡;(2)通過電子顯微鏡觀察微泡形態(tài);采用bicinchoninic acid(BCA)法標(biāo)定微泡數(shù)量;采用流式細(xì)胞儀檢測其表面標(biāo)志物;(3)將微泡與正常人外周血單個核細(xì)胞(peripheral blood mononuclear cells,PB-MNC)共培養(yǎng)12h后,通過共聚焦掃描電鏡觀察微泡與單個核細(xì)胞的作用方式;共培養(yǎng)48h后采用酶聯(lián)免疫吸附試驗法(ELISA)法檢測上清中IL-2,IL-6,IL-8,IL-10,IFN-γ及TNF-α的分泌量;共培養(yǎng)48h后采用流式細(xì)胞儀檢測T細(xì)胞亞群及T細(xì)胞激活變化;共培養(yǎng)48h后采用流式細(xì)胞儀檢測不同亞群細(xì)胞胞內(nèi)細(xì)胞因子染色情況;(4)采用甲基纖維素半固體培養(yǎng)基檢測微泡及微泡與單個核細(xì)胞共培養(yǎng)48小時后上清液對外周血造血干細(xì)胞集落形成的影響。研究結(jié)果通過密度梯度離心法可提取出動員后外周血造血干細(xì)胞。利用超速離心法可成功提取出外周血造血干細(xì)胞來源的微泡。透射電子顯微鏡觀察分離得到的微泡為卵圓形膜性小囊泡。流式細(xì)胞術(shù)測得微泡高表達(dá)其特異性標(biāo)志CD63(85.86%),并帶有其母體細(xì)胞標(biāo)志如干細(xì)胞標(biāo)志CD34(33.52%),T細(xì)胞標(biāo)志CD3(43.98%)、CD45RA(23.54%)、CD45RO(68.3%),NK細(xì)胞標(biāo)志CD56(32.32%)等。共聚焦掃描電鏡顯示微泡可以與外周血單個核細(xì)胞相融合并促進(jìn)其分泌IL-6,IL-8,IL-10與TNF-α細(xì)胞因子。流式細(xì)胞儀檢測結(jié)果顯示T細(xì)胞亞群及T細(xì)胞激活無明顯改變。胞內(nèi)因子染色結(jié)果表明CD4+,CD8+,CD11c+細(xì)胞內(nèi)因子無明顯改變。集落培養(yǎng)實驗表明微泡及微泡與單個核細(xì)胞共培養(yǎng)48小時后上清液很可能具有促進(jìn)造血集落形成的作用。研究結(jié)論外周血造血干細(xì)胞來源的微泡具有免疫調(diào)節(jié)及可能促進(jìn)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.

【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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