本文選題：脂肪組織　切入點(diǎn)：內(nèi)皮祖細(xì)胞　出處：《南方醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：背景:內(nèi)皮祖細(xì)胞(Endothelial progenitor cells,EPCs)是成熟內(nèi)皮細(xì)胞的前體細(xì)胞,可遷移到缺血組織,分化為成熟內(nèi)皮細(xì)胞(Enthothelial cells,ECs),發(fā)揮血管新生作用。隨著EPCs研究的深入,其在臨床診斷、預(yù)后判斷和各種缺血性疾病的治療方面將會有廣闊的應(yīng)用前景。EPCs不僅存在于骨髓、外周血和臍血中,還存在于胚胎、心臟、骨骼肌和血管中。然而,這些來源的EPCs都存在一定的限制。因此,尋找合適來源的EPCs就變得尤為重要。近年來的研究發(fā)現(xiàn),脂肪組織中含有多種細(xì)胞,包括EPCs。脂肪組織不僅具有來源豐富,獲取容易且其對人體創(chuàng)傷較小的優(yōu)勢,而且其種子細(xì)胞豐富,適合細(xì)胞的自體移植,因此,脂肪組織是理想的EPCs種子細(xì)胞來源,但目前國內(nèi)外缺乏有效的分離培養(yǎng)脂肪源性EPCs的方法。目的:探討一種有效、經(jīng)濟(jì)、可行的從人脂肪組織中分離培養(yǎng)EPCs的方法,并對其生物學(xué)特性展開研究。方法:來用酶消化法從人脂肪組織中分離培養(yǎng)出脂肪基質(zhì)血管細(xì)胞(Stromal vascular cells,SVF),流式細(xì)胞術(shù)檢測SVF的免疫表型以分析其細(xì)胞成分。通過EPCs和脂肪干細(xì)胞(Adipose stem cells,ASCs)對胰蛋白酶的敏感性不同,差速消化分離EPCs和ASCs。觀察細(xì)胞的形態(tài)特征,繪制細(xì)胞的生長曲線、計(jì)算細(xì)胞的細(xì)胞倍增數(shù)(PD)和倍增時(shí)間(DT)評估細(xì)胞的生長增殖能力,流式細(xì)胞分析術(shù)檢測EPCs的表型特征,免疫熒光染色觀察細(xì)胞攝取FITC-UEA-1和吞噬Dil-ac-LDL的能力。最后,通過體外成血管試驗(yàn)分析EPCs的血管形成能力。結(jié)果:通過差速消化分離法,我們成功從脂肪組織中分離出EPCs和ASCs。流式細(xì)胞術(shù)檢測分析顯示EPCs表達(dá)CD31、CD34和VEGFR2,而幾乎不表達(dá)造血干細(xì)胞表面標(biāo)志CD45;體外擴(kuò)增培養(yǎng)后呈典型的鋪路石樣形態(tài),并可吞噬乙�；兔芏戎鞍�(Dil-ac-LDL)和結(jié)合荊豆凝集素(FITC-UEA-1),在熒光顯微鏡下觀察吞噬Dil-ac-LDL的EPCs呈紅色熒光,而結(jié)合FITC-UEA-1呈綠色熒光。此外,將其接種于Matrigel人工基底膜,可形成血管腔樣的結(jié)構(gòu)。ASCs高度表達(dá)CD29、CD73、CD90、CD105等間充質(zhì)細(xì)胞表面標(biāo)志,體外擴(kuò)增培養(yǎng)呈長梭形纖維細(xì)胞樣生長。結(jié)論:我們通過差速消化分離法成功從人脂肪組織中分離培養(yǎng)出EPCs,為脂肪源性EPCs的分離培養(yǎng)提供了一種有效、經(jīng)濟(jì)、可行的研究方法,為各種缺血性疾病提供了豐富的種子細(xì)胞來源,給治療性血管新生和再生醫(yī)學(xué)提供了廣闊的應(yīng)用前景。
[Abstract]:Background: Endothelial progenitor cells (EPCs) are progenitor cells of mature endothelial cells, which can migrate to ischemic tissue and differentiate into endothelial cells of endothelial cells (ECs), which play a role in angiogenesis. Prognosis judgment and treatment of various ischemic diseases will have broad application prospects. EPCs exist not only in bone marrow, peripheral blood and umbilical cord blood, but also in embryo, heart, skeletal muscle and blood vessel. EPCs from these sources has certain limitations. Therefore, it is particularly important to find suitable sources of EPCs. Recent studies have found that adipose tissue contains a variety of cells, including EPCs.Adipose tissue is not only rich in sources. Therefore, adipose tissue is an ideal source of EPCs seed cells, because it is easy to obtain and has less trauma to human body, and its seed cells are abundant and suitable for autologous transplantation of cells. But there is a lack of effective method to isolate and culture adipose EPCs at home and abroad. Objective: to explore an effective, economical and feasible method for isolation and culture of EPCs from human adipose tissue. Methods: stromal vascular cells were isolated and cultured from human adipose tissue by enzyme digestion, and the immunophenotype of SVF was detected by flow cytometry. The cell components were analyzed by EPCs. The sensitivity to trypsin is different from adipose stem cells. EPCs and ASCs were separated by differential digestion. The morphological characteristics of cells were observed, cell growth curves were drawn, cell multiplication number and multiplication time were calculated to evaluate cell growth and proliferation ability, and phenotypic characteristics of EPCs were detected by flow cytometry. The ability of FITC-UEA-1 uptake and Dil-ac-LDL phagocytosis was observed by immunofluorescence staining. Finally, the angiogenesis ability of EPCs was analyzed by in vitro vascularization test. We successfully isolated EPCs and ASCs.FCM analysis from adipose tissue showed that EPCs expressed CD31mCD34 and VEGFR2, but hardly expressed CD45. after amplification and culture in vitro, we showed typical paving stone shape. EPCs phagocytosis of Dil-ac-LDL and green fluorescence of FITC-UEA-1 were observed under fluorescence microscope. In addition, it was inoculated into Matrigel artificial basement membrane. ASCs highly expressed CD29, CD73, CD90, CD105 and other mesenchymal cell surface markers. Conclusion: we successfully isolated and cultured EPCs from human adipose tissue by differential digestion, which provides an effective, economical and feasible method for the isolation and culture of adipose derived EPCs. It provides abundant seed cell sources for various ischemic diseases and provides a broad application prospect for therapeutic angiogenesis and regenerative medicine.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R329.2

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相關(guān)期刊論文 前1條

1 劉琴;王麗平;陳芳;張宜;;凍存SD大鼠脂肪組織中脂肪干細(xì)胞的分離培養(yǎng)和鑒定[J];細(xì)胞與分子免疫學(xué)雜志;2017年02期

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