發(fā)布時間：2019-04-03 19:44

【摘要】： 骨髓造血微環(huán)境(hematopoietic inductive microenvironment,HIM)是造血干/祖細(xì)胞生長發(fā)育的“土壤”,基質(zhì)細(xì)胞作為造血微環(huán)境中的重要成分,不僅與造血干/祖細(xì)胞的歸巢定位、增殖分化和自我更新密切相關(guān),而且在某些血液系統(tǒng)疾病的發(fā)生、發(fā)展和轉(zhuǎn)歸過程中具有非常重要的作用,探討基質(zhì)細(xì)胞在造血調(diào)控中的作用需繼續(xù)深入。 人臍血中的細(xì)胞成分豐富且較骨髓和外周血更原始;臍血細(xì)胞具有來源廣泛、采集方便、GVHD輕和高增殖的特點,臨床應(yīng)用前景廣闊。目前對造血基質(zhì)細(xì)胞的研究主要側(cè)重于骨髓基質(zhì)細(xì)胞,而對于臍血中是否存在基質(zhì)細(xì)胞,臍血源基質(zhì)細(xì)胞的生物學(xué)特點如何,能否作為一種新型的造血基質(zhì)細(xì)胞來源等問題尚缺乏系統(tǒng)深入的研究;血管細(xì)胞粘附分子(vascular cell adhsion molecule-1,VCAM-1)是骨髓基質(zhì)細(xì)胞參與造血調(diào)控不可缺少的粘附分子,它與受體整聯(lián)蛋白(Integrin)家族中的α4β1特異性結(jié)合能起到固定造血干細(xì)胞于骨髓基質(zhì)的作用,對造血干/祖細(xì)胞的增殖、分化、遷移中發(fā)揮重要調(diào)控作用。 鑒于此,本課題將人臍血CD34+細(xì)胞,采用Dexter貼壁細(xì)胞培養(yǎng)體系行臍血源基質(zhì)細(xì)胞培養(yǎng),探討人臍血細(xì)胞體外擴增基質(zhì)細(xì)胞的可行性和有效性;研究人臍血源基質(zhì)細(xì)胞微環(huán)境的造血支持作用;構(gòu)建熒光蛋白標(biāo)記的VCAM-1腺病毒載體并轉(zhuǎn)染人臍血源基質(zhì)細(xì)胞,移植給造血微環(huán)境輻射損傷裸鼠,探討其對重建造血微環(huán)境功能及促進造血損傷修復(fù)的作用,為平戰(zhàn)條件下造血功能損傷的救治提供新的輔助治療措施。 1.研究內(nèi)容及方法: 1.1分離人臍血CD34+細(xì)胞,采用Dexter貼壁細(xì)胞培養(yǎng)體系行臍血源基質(zhì)細(xì)胞培養(yǎng)、傳代擴增,采用光鏡,電鏡、組織細(xì)胞化學(xué)、免疫細(xì)胞化學(xué)和流式細(xì)胞儀等技術(shù)對臍血源基質(zhì)細(xì)胞進行鑒定; 1.2采用DNA重組技術(shù),將目的基因VCAM-1克隆至含有報告基因EGFP的穿梭質(zhì)粒;在BJ5183細(xì)胞中與腺病毒基因組進行同源重組,產(chǎn)生重組腺病毒;在脂質(zhì)體介導(dǎo)下,將重組的腺病毒表達質(zhì)粒轉(zhuǎn)染293細(xì)胞,使腺病毒在293細(xì)胞中包裝復(fù)制,并對轉(zhuǎn)染后的293細(xì)胞進行熒光觀測,檢測培養(yǎng)上清目的蛋白的表達;用高效轉(zhuǎn)染載
[Abstract]:Bone marrow hematopoietic microenvironment (hematopoietic inductive microenvironment,HIM) is the "soil" for the growth and development of hematopoietic stem / progenitor cells. As an important component of hematopoietic microenvironment, stromal cells are not only associated with homing and localization of hematopoietic stem / progenitor cells. Proliferation, differentiation and self-renewal are closely related to each other, and play an important role in the occurrence, development and prognosis of some hematological diseases. It is necessary to explore the role of stromal cells in hematopoiesis regulation. Human umbilical cord blood is rich in cellular components and more primitive than bone marrow and peripheral blood. Umbilical cord blood cells have a wide range of sources, convenient collection, light and high proliferation of GVHD, and have broad clinical application prospects. At present, the research on hematopoietic stromal cells mainly focuses on bone marrow stromal cells, but for whether there are stromal cells in umbilical cord blood, what are the biological characteristics of umbilical cord blood derived stromal cells? Whether it can be used as a new source of hematopoietic stromal cells and other issues is still lack of systematic and in-depth research; Vascular cell adhesion molecule (vascular cell adhsion molecule-1,VCAM-1) is an indispensable adhesion molecule for bone marrow stromal cells to participate in hematopoiesis regulation. Its specific binding to 偽 4 尾 1 in the receptor integrin (Integrin) family plays a role in fixing hematopoietic stem cells in bone marrow stroma and plays an important role in regulating the proliferation, differentiation and migration of hematopoietic stem / progenitor cells. In view of this, human umbilical cord blood CD34 cells were cultured with Dexter adherent cell culture system to explore the feasibility and effectiveness of expansion of stromal cells in vitro by human umbilical cord blood cells. To study the hematopoietic supporting effect of microenvironment of human umbilical cord blood-derived stromal cells (UCB). To construct VCAM- 1 adenovirus vector labeled with fluorescent protein and transfect human umbilical cord blood-derived stromal cells into nude mice with radiation-induced hematopoietic microenvironment injury, and to explore the effects of adenovirus vector labeled with fluorescent protein on the reconstruction of hematopoietic microenvironment and the repair of hematopoietic injury. To provide a new adjuvant therapy for the treatment of hematopoiesis injury in peacetime and war. 1. Content and methods: 1.1.1Human umbilical cord blood CD34 cells were isolated and cultured with Dexter adherent cell culture system. The cells were subcultured and amplified by light microscope, electron microscope, histochemistry, and the results were as follows: (1) Human umbilical cord blood derived stromal cells were cultured in vitro and cultured in vitro. Immunocytochemistry and flow cytometry were used to identify umbilical cord blood derived stromal cells. The target gene VCAM-1 was cloned into shuttle plasmid containing reporter gene EGFP by DNA recombination technique, and the recombinant adenovirus was generated by homologous recombination with adenovirus genome in BJ5183 cells. The recombinant adenovirus expression plasmid was transfected into 293 cells by lipofectamine, and the adenovirus was packaged and duplicated in 293 cells, and the transfected 293 cells were observed by fluorescence to detect the expression of the protein in the culture supernatant, and the recombinant adenovirus was transfected into 293 cells by high efficiency transfection.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2006
【分類號】：R329

【引證文獻】

相關(guān)博士學(xué)位論文 前1條

1 馮一梅;高表達SDF-1人臍血源基質(zhì)細(xì)胞經(jīng)PECAM-1介導(dǎo)調(diào)控巨核細(xì)胞增殖遷移的機制研究[D];第三軍醫(yī)大學(xué);2011年

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本文編號：2453507