缺氧態(tài)下Galectin-3對外周血內(nèi)皮祖細胞源性血管內(nèi)皮細胞的影響及機制研究
發(fā)布時間：2018-08-20 16:51
【摘要】：背景：血管發(fā)生曾被認為只存在于胚胎期原始血管網(wǎng)形成過程中。后證實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)生機制。傳統(tǒng)觀點認為,新生血管形成主要包括兩種機制,血管新生與血管發(fā)生：血管新生(angiogenesis)是指通過原存在血管的成熟內(nèi)皮細胞的增殖和遷移,以出芽方式長出新的毛細血管的過程,這一過程曾被認為是出生后血管形成的唯一機制,而血管發(fā)生(vasculogenesis)則是通過骨髓起源的內(nèi)皮祖細胞(EPCs)聚集到缺血損傷部位并分化為血管內(nèi)皮細胞(endothelialeens, ECs),并組織成血管的過程。在臨床應用中,干細胞(血管中EPCs)移植新生血管為治療缺血性疾病提供了另一個選擇。 研究目的：將人外周血內(nèi)皮祖細胞分離、培養(yǎng)并定向誘導分化為內(nèi)皮細胞,研究其體外誘導培養(yǎng)的條件及生物學活性,探索細胞生長最佳干預期,為人外周血EPCs的研究奠定基礎。同時,在缺氧態(tài)下觀察Galectin-3對內(nèi)皮祖細胞源性血管內(nèi)皮細胞增殖能力的影響,研究干細胞移植治療缺血性疾病的相關機制。 方法：本實驗研究主要分為三個方面：一、人外周血內(nèi)皮祖細胞的分離、培養(yǎng)、鑒定及活性研究：無菌條件下采集健康捐獻者外周靜脈血20ml,采用密度梯度離心法(Ficoll法)分離人外周血單個核細胞,通過細胞計數(shù),臺盼藍染色鑒定細胞活力后,將其接種在預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胞的生長形態(tài)學變化,觀測細胞生長情況,進行EPCs特征的鑒定：VEGFR-2、CD133、vWF的免疫熒光檢測；行細胞功能實驗：攝取acLDL和結合UEA-1檢測。 二、缺氧態(tài)下內(nèi)皮祖細胞的生長觀察：收集培養(yǎng)7天的內(nèi)皮祖細胞,隨機分配為氯化鈷(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,觀察在不同時間不同缺氧濃度下內(nèi)皮祖細胞的生長情況,MTT法觀察其生長情況,確定缺氧環(huán)境的最佳干預期及濃度。 三、缺氧態(tài)下Galectin-3對內(nèi)皮細胞增殖能力的影響：收集貼壁的成熟血管內(nèi)皮細胞細胞,在100umol/L氯化鈷濃度下,隨機分為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,MTT比色法觀察缺氧態(tài)下不同濃度Galectin-3對內(nèi)皮細胞增殖能力的影響。 結果：分離獲得的單個核細胞,細胞計數(shù)、臺盼藍染色后,活細胞百分率在(96±3)%,培養(yǎng)4天后絕大多數(shù)細胞貼壁生長,多為圓形或橢圓形；第5天起,部分較大圓形細胞轉(zhuǎn)化為梭狀內(nèi)皮樣細胞；第7天可見部分細胞聚集成集落；10天左右,細胞變?yōu)闂l索狀,相互交聯(lián)。在培養(yǎng)的第7天,細胞生長增殖情況最佳。對細胞性功能和特征測定,細胞的內(nèi)皮祖細胞表面特異性標志因子VEGFR-2、CD133與vWF表達情況尚佳,攝取acLDL和結合UEA-1檢測實驗結果顯示強陽性。細胞繼續(xù)培養(yǎng)約1月時可見細胞成典型鋪路石樣改變。隨著細胞培養(yǎng)液中CoCl2濃度的增加及培養(yǎng)時間的延長,細胞生長抑制率呈增加趨勢,其中CoCl2100μmol/L,培養(yǎng)24h細胞的生長情況較其他組好。Galectin-3在缺氧態(tài)下亦可明顯增加血管內(nèi)皮細胞增殖能力,呈一定的量效關系,其中5.0μg/ml和10μg/ml濃度的可明顯促進內(nèi)皮細胞的增殖能力(P0.05)。 結論：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)皮祖細胞,活細胞百分率在(96±3)%。2、在缺氧態(tài)下(CoCl2100μmol/L),細胞培養(yǎng)24h的生長情況最佳。3、在同一Galectin-3濃度下,缺氧態(tài)較常氧態(tài)對細胞增殖的促進作用更強。
[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
【學位授予單位】：昆明醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R329
本文編號：2194328
[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
【學位授予單位】：昆明醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R329
【參考文獻】
相關期刊論文 前2條
1 李珊姍;楊鏞;;Galectin-3對外周血內(nèi)皮祖細胞源性血管內(nèi)皮細胞增殖能力的影響[J];中國普外基礎與臨床雜志;2012年07期
2 楊國凱;楊鏞;何曉明;羅開元;萬嘉;楊光;李國劍;馬震寰;陸平;;外周血干細胞移植治療血栓閉塞性脈管炎[J];中國微創(chuàng)外科雜志;2009年09期
，本文編號：2194328
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