【摘要】：背景：血管發(fā)生曾被認(rèn)為只存在于胚胎期原始血管網(wǎng)形成過(guò)程中。后證實(shí)出生后的新血管形成并不僅限于血管新生,也包括胚胎期的血管發(fā)生機(jī)制。傳統(tǒng)觀點(diǎn)認(rèn)為,新生血管形成主要包括兩種機(jī)制,血管新生與血管發(fā)生：血管新生(angiogenesis)是指通過(guò)原存在血管的成熟內(nèi)皮細(xì)胞的增殖和遷移,以出芽方式長(zhǎng)出新的毛細(xì)血管的過(guò)程,這一過(guò)程曾被認(rèn)為是出生后血管形成的唯一機(jī)制,而血管發(fā)生(vasculogenesis)則是通過(guò)骨髓起源的內(nèi)皮祖細(xì)胞(EPCs)聚集到缺血損傷部位并分化為血管內(nèi)皮細(xì)胞(endothelialeens, ECs),并組織成血管的過(guò)程。在臨床應(yīng)用中,干細(xì)胞(血管中EPCs)移植新生血管為治療缺血性疾病提供了另一個(gè)選擇。 研究目的：將人外周血內(nèi)皮祖細(xì)胞分離、培養(yǎng)并定向誘導(dǎo)分化為內(nèi)皮細(xì)胞,研究其體外誘導(dǎo)培養(yǎng)的條件及生物學(xué)活性,探索細(xì)胞生長(zhǎng)最佳干預(yù)期,為人外周血EPCs的研究奠定基礎(chǔ)。同時(shí),在缺氧態(tài)下觀察Galectin-3對(duì)內(nèi)皮祖細(xì)胞源性血管內(nèi)皮細(xì)胞增殖能力的影響,研究干細(xì)胞移植治療缺血性疾病的相關(guān)機(jī)制。 方法：本實(shí)驗(yàn)研究主要分為三個(gè)方面：一、人外周血內(nèi)皮祖細(xì)胞的分離、培養(yǎng)、鑒定及活性研究：無(wú)菌條件下采集健康捐獻(xiàn)者外周靜脈血20ml,采用密度梯度離心法(Ficoll法)分離人外周血單個(gè)核細(xì)胞,通過(guò)細(xì)胞計(jì)數(shù),臺(tái)盼藍(lán)染色鑒定細(xì)胞活力后,將其接種在預(yù)先包被有人纖維連接蛋白的培養(yǎng)皿中培養(yǎng)。每日于倒置相差顯微鏡下觀察細(xì)胞的生長(zhǎng)形態(tài)學(xué)變化,觀測(cè)細(xì)胞生長(zhǎng)情況,進(jìn)行EPCs特征的鑒定：VEGFR-2、CD133、vWF的免疫熒光檢測(cè)；行細(xì)胞功能實(shí)驗(yàn)：攝取acLDL和結(jié)合UEA-1檢測(cè)。 二、缺氧態(tài)下內(nèi)皮祖細(xì)胞的生長(zhǎng)觀察：收集培養(yǎng)7天的內(nèi)皮祖細(xì)胞,隨機(jī)分配為氯化鈷(CoCl2)濃度分別為0,50,75,100,150,200,300μmol/L的濃度組,分別培養(yǎng)6h,12h,24h,48h,72h,觀察在不同時(shí)間不同缺氧濃度下內(nèi)皮祖細(xì)胞的生長(zhǎng)情況,MTT法觀察其生長(zhǎng)情況,確定缺氧環(huán)境的最佳干預(yù)期及濃度。 三、缺氧態(tài)下Galectin-3對(duì)內(nèi)皮細(xì)胞增殖能力的影響：收集貼壁的成熟血管內(nèi)皮細(xì)胞細(xì)胞,在100umol/L氯化鈷濃度下,隨機(jī)分為Galectin-3終濃度各濃度組(共6組)：在培養(yǎng)液中加入終濃度為0u g/ml,1μg/ml,2.5μg/ml,5μ g/ml,10μg/ml,15μg/ml Galectin-3培養(yǎng)24小時(shí),MTT比色法觀察缺氧態(tài)下不同濃度Galectin-3對(duì)內(nèi)皮細(xì)胞增殖能力的影響。 結(jié)果：分離獲得的單個(gè)核細(xì)胞,細(xì)胞計(jì)數(shù)、臺(tái)盼藍(lán)染色后,活細(xì)胞百分率在(96±3)%,培養(yǎng)4天后絕大多數(shù)細(xì)胞貼壁生長(zhǎng),多為圓形或橢圓形；第5天起,部分較大圓形細(xì)胞轉(zhuǎn)化為梭狀內(nèi)皮樣細(xì)胞；第7天可見(jiàn)部分細(xì)胞聚集成集落；10天左右,細(xì)胞變?yōu)闂l索狀,相互交聯(lián)。在培養(yǎng)的第7天,細(xì)胞生長(zhǎng)增殖情況最佳。對(duì)細(xì)胞性功能和特征測(cè)定,細(xì)胞的內(nèi)皮祖細(xì)胞表面特異性標(biāo)志因子VEGFR-2、CD133與vWF表達(dá)情況尚佳,攝取acLDL和結(jié)合UEA-1檢測(cè)實(shí)驗(yàn)結(jié)果顯示強(qiáng)陽(yáng)性。細(xì)胞繼續(xù)培養(yǎng)約1月時(shí)可見(jiàn)細(xì)胞成典型鋪路石樣改變。隨著細(xì)胞培養(yǎng)液中CoCl2濃度的增加及培養(yǎng)時(shí)間的延長(zhǎng),細(xì)胞生長(zhǎng)抑制率呈增加趨勢(shì),其中CoCl2100μmol/L,培養(yǎng)24h細(xì)胞的生長(zhǎng)情況較其他組好。Galectin-3在缺氧態(tài)下亦可明顯增加血管內(nèi)皮細(xì)胞增殖能力,呈一定的量效關(guān)系,其中5.0μg/ml和10μg/ml濃度的可明顯促進(jìn)內(nèi)皮細(xì)胞的增殖能力(P0.05)。 結(jié)論：1、采用Ficoll法從人外周血中可以成功分離出具有增殖分化潛能的內(nèi)皮祖細(xì)胞,活細(xì)胞百分率在(96±3)%。2、在缺氧態(tài)下(CoCl2100μmol/L),細(xì)胞培養(yǎng)24h的生長(zhǎng)情況最佳。3、在同一Galectin-3濃度下,缺氧態(tài)較常氧態(tài)對(duì)細(xì)胞增殖的促進(jìn)作用更強(qiáng)。
[Abstract]:BACKGROUND: Angiogenesis was once thought to exist only in the process of embryonic primitive angiogenesis. It has been proved that postnatal angiogenesis is not limited to angiogenesis, but also includes the mechanism of embryonic angiogenesis. Genesis is the process by which new capillaries are sprouted by the proliferation and migration of mature endothelial cells that originate from blood vessels. This process was once thought to be the only mechanism of post-natal angiogenesis, whereas vasculogenesis is the accumulation of bone marrow-derived endothelial progenitor cells (EPCs) into ischemic sites. Endothelial cells (ECs) differentiate into vascular endothelial cells (ECs) and organize the process of angiogenesis. In clinical applications, stem cells (EPCs) transplantation of new blood vessels provides another option for the treatment of ischemic diseases.
OBJECTIVE: To isolate, culture and differentiate human peripheral blood EPCs into endothelial cells (EPCs), to study the conditions and biological activities of EPCs in vitro, and to explore the optimal intervening period of EPCs in human peripheral blood. The effect of stem cell transplantation on ischemic disease is studied.
Methods: This study was divided into three aspects: 1. Isolation, culture, identification and activity of human peripheral blood endothelial progenitor cells (EPCs): 20 ml peripheral venous blood from healthy donors was collected under aseptic conditions, and human peripheral blood mononuclear cells were isolated by density gradient centrifugation (Ficoll method). The cells were identified by cell count and trypan blue staining. After viability, the cells were cultured in a dish pre-coated with human fibronectin. The morphological changes of the cells were observed daily under an inverted phase contrast microscope, and the growth of the cells was observed. Test.
2. Growth observation of EPCs under hypoxia condition: EPCs were collected and cultured for 7 days and randomly assigned to the concentration groups of 0,50,75,100,150,200,300 micromol/L of cobalt chloride (CoCl2). The EPCs were cultured for 6 h, 12 h, 24 h, 48 h and 72 h respectively. The growth of EPCs was observed by MTT method. The best dry anticipation and concentration of anoxic environment were determined.
3. Effects of Galectin-3 on the proliferation of endothelial cells under hypoxia: Adherent mature vascular endothelial cells were collected and randomly divided into six groups at 100 umol/L of cobalt chloride concentration. The final concentration of Galectin-3 was 0 u g/ml, 1 ug/ml, 2.5 ug/ml, 5 ug/ml, 10 ug/ml, 15 ug/ml of Galectin-3 was added into the culture medium. 24 hours, MTT colorimetric method was used to observe the effect of different concentrations of Galectin-3 on endothelial cell proliferation under hypoxia.
Results: Mononuclear cells were isolated and counted. After trypan blue staining, the percentage of living cells was (96 6550 Cells grew and proliferated best on the 7th day of culture. The expression of endothelial progenitor cell surface-specific markers, such as VEGFR-2, CD133 and vWF, was still good. The results of acLDL uptake and UEA-1 binding assay showed that the cells were strongly positive. With the increase of the concentration of CoCl2 in the culture medium and the prolongation of the culture time, the growth inhibition rate of the cells increased. The growth of the cells cultured for 24 hours was better than that of the other groups. Galectin-3 could also significantly increase the proliferation ability of vascular endothelial cells under hypoxia. The 5 g/ml and 10 g/ml concentrations significantly promoted the proliferation of endothelial cells (P0.05).
CONCLUSION: 1. Endothelial progenitor cells with proliferative and differentiative potential can be successfully isolated from human peripheral blood by Ficoll method. The percentage of living cells is (96 65
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R329

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本文編號(hào)：2194328