不同浓度EGF对兔ADSCs体外诱导分化为表皮细胞的实验研究

摘要

Objective To explore the EGF on ADSCs in rabbits in vitro differentiation of epidermal cells and the best concentration for tissue engineering on plastic surgery research provides theoretical basis for further .Explore the EGF on ADSCs in rabbits invitro differentiate of epidermal cells as well as the best optimal concentration needed for tissue engineering to provide further theoretical basis in plastic surgery.Methods Fat isolated from 2 months healthy white rabbit was isolated,cultured,generated ADSCs. Experiment is divided into induction group and not induced group,induced group with 8ng/ml,10ng/ml,20ng/ml of EGF to intervene,not induced group were compared with the same amount of saline.The work also includes observing the different concentration of EGF on ADSCs to epidermis cells in the change of morphology;ADSCs was induced 1 week and detection the expression and of CK-19,and Fluorescence PCR for testing the expression of CK-19 and integrinβ mRNA expression.ADSCs induced by one week of cells by immunofluorescence CK-19 expression was detected,and the lfuorescence quantitative PCR to detect CK-19 integrin β of mRNA expression.Results Induction of groups with different concentration of EGF was not obvious change in morphology.The shape were still a long fusiform or polygon; Immunolfuorescence showed that some cells of EGF for 10ng/ml can be induced group positive expression of the epidermal cells specific markers CK-19 and the number of expression is significantly higher than other groups,not induced group of cells in the test were negative; Fluorescence quantitative PCR results showed that the group of EGF for 10ng/ml expressing speciifcity factor CK-19 and mRNA of integrinβ were signiifcantly higher than the rest of the group (P<0.01).Conclusion EGF can successfully induced ADSCs to epidermal cells.Different concentrations of EGF on the source of ADSCs in rabbit in vitro differentiation,the best concentration of EGF is 10ng/ml.%目的：探究表皮生长因子（EGF）对兔脂肪来源干细胞（ADSCs）体外诱导分化为表皮细胞以及所需最佳浓度的实验研究，为组织工程在大面积皮肤缺损方面的研究提供进一步的理论依据。方法：取2个月龄健康新西兰大白兔的脂肪组织，体外分离、培养并传代ADSCs。实验分为诱导组和未诱导组，诱导组分别用5ng/ml、10ng/ml、20ng/ml的EGF进行干预，未诱导组用等量的生理盐水作空白对照。观察不同浓度的EGF对ADSCs诱导分化为表皮细胞的形态学变化，并于1周后对细胞进行免疫荧光及荧光定量PCR检测CK-19和整合素β（integrinβ）的表达情况。结果：光镜下显示诱导组细胞形态仍呈长梭形或多角形；免疫荧光结果显示诱导组（10ng/ml组）表皮细胞特异性标记物CK-19表达呈阳性，且其表达量明显高于其他组，未诱导组细胞呈阴性；荧光定量PCR结果显示诱导组（10ng/ml组）表皮细胞的特异性因子CK-19和integrinβ的mRNA表达量均明显高于其余组（P＜0.01）。结论：表皮生长因子能够成功诱导兔脂肪来源干细胞定向分化为表皮细胞，且其最佳诱导浓度为10ng/ml。