本文選題：內(nèi)皮祖細(xì)胞 + 血管新生　； 參考：《吉林大學(xué)》2011年碩士論文

【摘要】：當(dāng)機體發(fā)生缺血性腦卒中時,血管損傷是重要的病理基礎(chǔ)。目前治療均不能完全恢復(fù)血管的解剖結(jié)構(gòu),恢復(fù)腦卒中后缺血半暗帶及壞死區(qū)的血液供應(yīng),促進(jìn)缺血區(qū)血管生長的意義更為重大。血管新生和血管發(fā)生是血管生長的兩種主要形式。血管新生即在已有的血管處芽生出新的毛細(xì)血管。血管發(fā)生是在無血管區(qū)形成新的血管,即EPCs選擇性募集到受損或缺血組織區(qū)形成血管。機體發(fā)生缺血缺氧時,兩種成血管方式相互配合促進(jìn)新生血管形成。EPCs在缺血性疾病領(lǐng)域的研究已經(jīng)越來越多,本實驗旨在從大鼠骨髓及外周血中分離出EPCs并對其進(jìn)行鑒定,發(fā)現(xiàn)其特點及區(qū)別。 實驗使用密度梯度離心及貼壁篩選法從大鼠骨髓和外周血中分離獲得單個核細(xì)胞(MNC),進(jìn)行誘導(dǎo)培養(yǎng),觀察并記錄貼壁細(xì)胞的生物學(xué)特征；選取EPCs特異性表面標(biāo)志CD133、CD34、KDR對原代細(xì)胞進(jìn)行免疫熒光檢測,利用流式細(xì)胞學(xué)技術(shù)檢測KDR、CD34,并通過吞噬功能實驗進(jìn)一步鑒定培養(yǎng)細(xì)胞。 實驗結(jié)果： (1)大鼠骨髓和外周血能夠分離獲得內(nèi)皮祖細(xì)胞； (2)骨髓及外周血EPCs的生長共性：1.貼壁延遲性2.多種細(xì)胞形態(tài)：圓形細(xì)胞、長梭形細(xì)胞,有中央聚集成簇的圓形細(xì)胞和周邊放射狀細(xì)胞組成的“血島”樣細(xì)胞集落、條索樣、管狀、鵝卵石樣； (3)骨髓及外周血EPCs的生長異同：1、骨髓細(xì)胞集落數(shù)量多于外周血細(xì)胞集落數(shù)量；2、細(xì)胞培養(yǎng)前3d骨髓中單個核細(xì)胞貼壁明顯較外周血中多,但雜細(xì)胞較多；3、骨髓中EPCs細(xì)胞活性較強,增殖能力強,可見大量空腔樣或管腔樣,橋接樣細(xì)胞生長,可以大量傳代、凍存及復(fù)蘇。外周血中EPCs活性略差,一般生長4周后逐漸出現(xiàn)大量細(xì)胞變圓凋亡。 (4)貼壁細(xì)胞免疫熒光檢測CD34、CD133、KDR表達(dá)陽性； (5)流式細(xì)胞學(xué)檢測CD34、KDR表達(dá)陽性,陽性率分別為骨髓69.0%、84.8%,外周血5.4%、32.7%。 (6)細(xì)胞能夠吞噬ac-LDL,結(jié)合UEA-1,說明所獲細(xì)胞為功能完整的EPCs。 EPCs是一種能直接分化為血管內(nèi)皮細(xì)胞的前體細(xì)胞。它不僅參與了胚胎早期血管的形成,更是成年個體中與血管新生和血管形成關(guān)系最為密切的干細(xì)胞,在血管修復(fù)及側(cè)支血管網(wǎng)的建立中具有重要作用,具有促進(jìn)新生血管形成、參與缺血組織血管再生、促進(jìn)神經(jīng)再生、預(yù)測腦缺血發(fā)展及預(yù)后等功能。 近年來, EPCs移植應(yīng)用于血管相關(guān)性疾病的治療,利用EPCs作為種子細(xì)胞對血管新生的作用更加顯著,聯(lián)合基因治療效果更佳。我們以臨床應(yīng)用為目的,在動物實驗的基礎(chǔ)上,選取來源大鼠骨髓及外周血提取并培養(yǎng)EPCs,探尋出了穩(wěn)定、實用的EPCs體外分離、培養(yǎng)和鑒定的方法,探尋EPCs的最佳擴增體系以提供足量的有活力的可用于臨床的細(xì)胞源。
[Abstract]:Vascular injury is an important pathological basis when ischemic stroke occurs.Angiogenesis and angiogenesis are two main forms of angiogenesis.Angiogenesis is the formation of new capillaries in the bud of an existing vessel.Angiogenesis is the formation of new blood vessels in the absence of blood vessels, that is, the selective recruitment of EPCs to the damaged or ischemic tissue regions to form blood vessels.In the process of ischemia and hypoxia, there have been more and more researches on how to promote neovascularization. EPCs are isolated and identified from bone marrow and peripheral blood of rats.Find its characteristics and differences.The mononuclear cells were isolated from rat bone marrow and peripheral blood by density gradient centrifugation and adherent screening. The mononuclear cells were induced and cultured. The biological characteristics of adherent cells were observed and recorded.The EPCs specific surface marker CD133G CD34KDR was selected to detect the primary cells by immunofluorescence, and the KDRN CD34 was detected by flow cytometry, and the cultured cells were further identified by phagocytosis assay.Experimental results:1) Endothelial progenitor cells were isolated from rat bone marrow and peripheral blood.The growth commonness of EPCs in bone marrow and peripheral blood was 1: 1.Adhesion delay 2.A variety of cell morphology: round cells, long fusiform cells, there are central clusters of round cells and peripheral radial cells composed of "blood island" like cell colony, like, tubular, cobblestone;(3) the growth similarities and differences of EPCs in bone marrow and peripheral blood were observed. The number of bone marrow cells was more than that of peripheral blood cells. The number of mononuclear cells in bone marrow was significantly more than that in peripheral blood 3 days before cell culture, but the number of miscellaneous cells was more than that in peripheral blood, and the activity of EPCs cells in bone marrow was stronger.The proliferation ability is strong, a large number of cavity or tube cavity, bridging like cell growth, can be a large number of passage, cryopreservation and recovery.The activity of EPCs in peripheral blood was slightly poor, and a large number of cells became round and apoptotic gradually after 4 weeks of growth.The positive expression of KDR was detected by immunofluorescence in adherent cells.The positive rate of CD34 KDR expression was 69.0% in bone marrow and 84.8% in bone marrow, and 5.44% and 32.7% in peripheral blood respectively.(6) the cells could phagocytosis ac-LDL and combined with UEA-1, which indicated that the cells obtained were EPCs with complete function.EPCs is a kind of progenitor cells that can directly differentiate into vascular endothelial cells.Participate in vascular regeneration of ischemic tissue, promote nerve regeneration, predict the development and prognosis of cerebral ischemia.For the purpose of clinical application, on the basis of animal experiments, EPCs were extracted and cultured from rat bone marrow and peripheral blood, and a stable and practical method for isolation, culture and identification of EPCs in vitro was found.To explore the best amplification system of EPCs to provide a sufficient number of viable cell sources for clinical use.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

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相關(guān)期刊論文 前2條

1 鄭奇軍;劉維永;易定華;俞世強;劉洋;歐陽輝;程亮;蔡振杰;;骨髓內(nèi)皮祖細(xì)胞EPCs兩種分離方法及其對比研究[J];第四軍醫(yī)大學(xué)學(xué)報;2008年04期

2 朱軍慧;馬彩艷;陳君柱;王興祥;張芙榮;孫堅;;高同型半胱氨酸血癥患者循環(huán)內(nèi)皮祖細(xì)胞的變化[J];臨床心血管病雜志;2008年04期

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