本文選題：兔角膜緣干細(xì)胞 + 體外培養(yǎng)�。� 參考：《吉林大學(xué)》2007年碩士論文

【摘要】： 目的:通過體外培養(yǎng)兔角膜緣干細(xì)胞,觀察其生物學(xué)特性,建立兔角膜緣干細(xì)胞的體外培養(yǎng)方法。探討生長因子(EGF、bFGF、NGF)對兔角膜緣干細(xì)胞的增殖作用影響及最佳有效濃度。 方法:0.25%胰蛋白酶消化角膜緣組織,用含15%胎牛血清的DMEM:F12(1:1)(W/W)的培養(yǎng)液(DF)對兔角膜緣干細(xì)胞進(jìn)行體外培養(yǎng),形態(tài)學(xué)觀察,培養(yǎng)的細(xì)胞早期使用AE1/AE3(針對一組酸性和堿性角蛋白的抗體)、晚期使用AE5(對64KD角蛋白特異的單克隆抗體)作細(xì)胞免疫化學(xué)鑒定。取第2代生長狀態(tài)良好的干細(xì)胞,以5×103個(gè)ml細(xì)胞濃度接種于96孔細(xì)胞培養(yǎng)板中,依次加入不同濃度的EGF、bFGF及NGF培養(yǎng)48h后,采用MTT法測定細(xì)胞增殖情況。 結(jié)果:原代培養(yǎng)細(xì)胞48h后開始貼壁,部分細(xì)胞由圓形變?yōu)槁褕A形或長梭形;10～14d形成單層,細(xì)胞呈圓形、卵圓形,類角膜上皮細(xì)胞;細(xì)胞傳到第5代左右開始出現(xiàn)老化狀態(tài);免疫細(xì)胞化學(xué)染色:培養(yǎng)的細(xì)胞早期AE1/AE3呈陽性而少部分細(xì)胞AE5呈陽性,培養(yǎng)的細(xì)胞晚期AE5呈陽性。EGF濃度為10ng/ml、bFGF濃度為20ng/ml及NGF濃度為100ng/ml時(shí)對兔角膜緣干細(xì)胞的促增殖作用最佳(P0.01)。 結(jié)論:通過本文的實(shí)驗(yàn)方法,建立了兔角膜緣干細(xì)胞的體外培養(yǎng)方法。EGF、bFGF及NGF可有效促進(jìn)兔角膜緣干細(xì)胞的生長,培養(yǎng)的適宜濃度分別為10ng/ml、20ng/ml、100ng/ml。
[Abstract]:Aim: to establish a culture method of rabbit limbal stem cells by observing its biological characteristics in vitro.To investigate the effect of EGF- bFGF- NGF on the proliferation of rabbit limbal stem cells (limbus cornea) and its optimal effective concentration.Methods Corneal limbus tissue was digested with 0.25% trypsin and cultured in vitro with DMEM: F121W / W (DMEM: F121W) medium containing 15% fetal bovine serum. The corneal limbal stem cells were cultured in vitro and morphologically observed.The cultured cells were identified by immunocytochemistry with AE1 / AE3 (for a group of acidic and basic keratin antibodies) and AE5 (monoclonal antibody specific for 64KD keratin).The stem cells in good growth state of the second generation were inoculated in 96-well cell culture plate with 5 脳 103 ml cell concentration. The proliferation of the cells was measured by MTT method after 48 hours of culture with different concentrations of EGFN bFGF and NGF.Results: the primary cultured cells began to adhere to the wall after 48 hours, and some cells formed monolayer from round to oval or fusiform for 1014 days, the cells were round, oval, corneal epithelium cells, the cells reached to the fifth generation and began to appear aging state.Immunocytochemical staining showed that AE1/AE3 was positive in the early stage of cultured cells and AE5 was positive in a small number of cells. In the late stage of cultured cells, the concentration of AE5 was 10 ng / ml ~ (-1) 20ng/ml and the concentration of NGF was 100ng/ml. The best effect on proliferation of rabbit limbal stem cells was P0.01 ~ (-1).Conclusion: the in vitro culture methods of rabbit limbal stem cells. EGF- bFGF and NGF can effectively promote the growth of rabbit limbal stem cells. The suitable concentration of cultured limbal stem cells is 10 ng / ml ~ 20 ng / ml ~ 100 ng 路ml ~ (-1) 路L ~ (-1) 路L ~ (-1) 路L ~ (-1) 路L ~ (-1) 路L ~ (-1) 路L ~ (-1).
【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】：2007
【分類號】：R329

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