本文選題：單個(gè)核細(xì)胞 + 內(nèi)皮祖細(xì)胞�。� 參考：《鄭州大學(xué)》2010年碩士論文

【摘要】：研究背景和目的 內(nèi)皮祖細(xì)胞(endothelial progenitor cells, EPCs)是一種多能干細(xì)胞,它能自我更新并且增殖分化為成熟的血管內(nèi)皮細(xì)胞。它不但參與了胚胎發(fā)育早期的血管形成,在成年體內(nèi)與血管新生及血管形成關(guān)系也最為密切。此外,EPCs還可以分泌多種促進(jìn)新生血管生長(zhǎng)的細(xì)胞因子,例如表皮生長(zhǎng)因子(epidermal growth factor, EGF),成纖維細(xì)胞生長(zhǎng)因子4,9(fibroblast growth factor-4,9, FGF-4,9),血管內(nèi)皮生長(zhǎng)因子(vascular endothelial growth factor, VEGF)等等。近年來,EPCs在促進(jìn)受損血管的修復(fù)方面,如心腦血管疾病、糖尿病性視網(wǎng)膜病變及創(chuàng)傷愈合等多種血管類疾病的治療中展現(xiàn)出極為廣泛的應(yīng)用前景。建立獲取外周血EPCs的方法,將會(huì)為其作為種子細(xì)胞廣泛應(yīng)用于血管組織工程奠定基礎(chǔ)。 方法 本實(shí)驗(yàn)采用密度梯度離心法從大鼠外周血分離單個(gè)核細(xì)胞,按不同培養(yǎng)條件分成4組：1組：未鋪纖維連接蛋白,使用M199基本培養(yǎng)基(含M199培養(yǎng)基,10%胎牛血清)；2組：未鋪纖維連接蛋白,使用M199誘導(dǎo)培養(yǎng)基(含M199培養(yǎng)基,10%胎牛血清,血管內(nèi)皮生長(zhǎng)因子10ng/mL,堿性成纖維細(xì)胞生長(zhǎng)因子bFGF4ng/mL);3組：預(yù)鋪纖維連接蛋白(fibronectin,FN),使用M199基本培養(yǎng)基；4組：預(yù)鋪纖維連接蛋白,使用M199誘導(dǎo)培養(yǎng)基。分別對(duì)其進(jìn)行體外誘導(dǎo)培養(yǎng),比較其體外生長(zhǎng)情況的不同,應(yīng)用統(tǒng)計(jì)學(xué)軟件比較細(xì)胞培養(yǎng)第7天細(xì)胞數(shù)量、集落數(shù)有無統(tǒng)計(jì)學(xué)差異,繪制各組生長(zhǎng)曲線,并進(jìn)行免疫組化染色及免疫熒光的鑒定。 結(jié)果 大鼠外周血來源的單個(gè)核細(xì)胞在體外呈現(xiàn)貼壁生長(zhǎng),最初培養(yǎng)3到4天內(nèi)細(xì)胞生長(zhǎng)較慢,約4天后出現(xiàn)對(duì)數(shù)生長(zhǎng)期,10天后細(xì)胞基本長(zhǎng)滿培養(yǎng)瓶底。培養(yǎng)第7天各組細(xì)胞數(shù)及細(xì)胞集落數(shù)提示:在相同培養(yǎng)條件下(分別將第1組與第3組、第2組與第4組相比較),預(yù)鋪纖維連接蛋白可以促進(jìn)EPCs的貼壁增殖(t=4.43,P0.05；t=3.70,P0.05)。而在同樣未鋪或預(yù)鋪纖維連接蛋白的情況下(分別將1組與2組、3組與4組比較),在培養(yǎng)液中加入生長(zhǎng)因子可以促使單個(gè)核細(xì)胞更好地向EPCs分化,說明生長(zhǎng)因子對(duì)EPCs原代靶細(xì)胞有明顯的促增殖作用(t=—13.22,P0.01；t=—10.96,P0.01)。培養(yǎng)所得細(xì)胞表面CD34、Flk-1和CD133在不同時(shí)段以不同比率呈陽(yáng)性表達(dá)。 結(jié)論 從大鼠外周血中經(jīng)密度梯度離心法分離單個(gè)核細(xì)胞,進(jìn)行誘導(dǎo)培養(yǎng),可以獲得EPCs。纖維連接蛋白有利于EPCs的貼壁生長(zhǎng)和增殖。血管內(nèi)皮生長(zhǎng)因子及成纖維細(xì)胞生長(zhǎng)因子促進(jìn)其增殖分化。EPCs的體外成功培養(yǎng)將為其應(yīng)用于血管組織工程提供足夠數(shù)量的種子細(xì)胞來源,并為外周血干細(xì)胞移植治療多種疾病提供新的思路。
[Abstract]:Background and purpose of the study Endothelial progenitor cells (progenitor cells, EPCs) are pluripotent stem cells, which can self-renew and proliferate and differentiate into mature vascular endothelial cells. It is not only involved in angiogenesis in early embryonic development, but also closely related to angiogenesis and angiogenesis in adults. In addition, EPCs can also secrete a variety of cytokines that promote angiogenesis, such as epidermal growth factor, fibroblast growth factor-4, FGF-4, vascular endothelial growth factor endothelial growth factor, VEGF) and so on. In recent years, EPCs have been widely used in the treatment of vascular diseases such as cardiovascular and cerebrovascular diseases, diabetic retinopathy and wound healing. The method of obtaining EPCs from peripheral blood will lay a foundation for its wide application as seed cells in vascular tissue engineering. Method Mononuclear cells were isolated from the peripheral blood of rats by density gradient centrifugation. The mononuclear cells were divided into 4 groups according to different culture conditions: group 1: uncoated fibronectin, Using M199 basic medium (containing M199 medium / 10% fetal bovine serum): group 2: uncoated fibronectin, using M199 induction medium (M199 medium containing 10% fetal bovine serum), Vascular endothelial growth factor (10 ng / mL), basic fibroblast growth factor (bFGF4ng / mLL) group 3: precoated fibronectin FNN group, M199 basic medium: precoated fibronectin group 4: precoated fibronectin group, induced by M199 medium. The cell number and colony number were compared by statistical software on the 7th day of cell culture, and the growth curves of each group were drawn. Immunohistochemical staining and immunofluorescence were performed. Result The mononuclear cells from peripheral blood of rats showed adherent growth in vitro. The growth of mononuclear cells was slower in the first 3 to 4 days, and the logarithmic growth phase appeared after 4 days. After 10 days of logarithmic growth, the cells grew to the bottom of the culture flask. The cell number and colony number of each group on the 7th day of culture indicated that under the same culture conditions (group 1 and group 3, group 2 and group 4, respectively), the precoated fibronectin could promote the proliferation of adherent cells of EPCs. In the same condition without or without fibronectin (group 1 and group 2, group 3 and group 4, respectively), the addition of growth factor in the culture medium could promote the better differentiation of mononuclear cells into EPCs. The results indicated that the growth factor had obvious effect on the proliferation of EPCs primary target cells. CD34 flk-1 and CD133 were expressed at different rates on the surface of cultured cells. Conclusion Mononuclear cells were isolated from rat peripheral blood by density gradient centrifugation. Fibronectin is beneficial to the adherent growth and proliferation of EPCs. The successful culture of vascular endothelial growth factor and fibroblast growth factor to promote proliferation and differentiation of EPCs in vitro will provide a sufficient number of seed cell sources for its application in vascular tissue engineering. It also provides a new idea for peripheral blood stem cell transplantation in the treatment of various diseases.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

【共引文獻(xiàn)】

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本文編號(hào)：1975776