兔角膜缘干细胞的体外培养及生长因子对其增殖的影响

发布时间：2018-04-17 17:40

本文选题：兔角膜缘干细胞 + 体外培养　；参考：《吉林大学》2007年硕士论文

【摘要】： 目的:通过体外培养兔角膜缘干细胞,观察其生物学特性,建立兔角膜缘干细胞的体外培养方法。探讨生长因子(EGF、bFGF、NGF)对兔角膜缘干细胞的增殖作用影响及最佳有效浓度。方法:0.25%胰蛋白酶消化角膜缘组织,用含15%胎牛血清的DMEM:F12(1:1)(W/W)的培养液(DF)对兔角膜缘干细胞进行体外培养,形态学观察,培养的细胞早期使用AE1/AE3(针对一组酸性和碱性角蛋白的抗体)、晚期使用AE5(对64KD角蛋白特异的单克隆抗体)作细胞免疫化学鉴定。取第2代生长状态良好的干细胞,以5×103个ml细胞浓度接种于96孔细胞培养板中,依次加入不同浓度的EGF、bFGF及NGF培养48h后,采用MTT法测定细胞增殖情况。结果:原代培养细胞48h后开始贴壁,部分细胞由圆形变为卵圆形或长梭形;10～14d形成单层,细胞呈圆形、卵圆形,类角膜上皮细胞;细胞传到第5代左右开始出现老化状态;免疫细胞化学染色:培养的细胞早期AE1/AE3呈阳性而少部分细胞AE5呈阳性,培养的细胞晚期AE5呈阳性。EGF浓度为10ng/ml、bFGF浓度为20ng/ml及NGF浓度为100ng/ml时对兔角膜缘干细胞的促增殖作用最佳(P0.01)。结论:通过本文的实验方法,建立了兔角膜缘干细胞的体外培养方法。EGF、bFGF及NGF可有效促进兔角膜缘干细胞的生长,培养的适宜浓度分别为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).
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329

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