角质形成细胞、维生素C和胰岛素对成纤维细胞增殖的影响以及纤维膜的形成
发布时间：2018-04-19 15:24
本文选题：角质形成细胞 + 成纤维细胞 ； 参考：《苏州大学》2010年硕士论文
【摘要】： 目的:通过研究角质形成细胞、维生素C和胰岛素对成纤维细胞增殖的影响以及维生素C和胰岛素对成纤维细胞体外形成纤维膜的作用,为今后构建不含异种胶原的真皮替代物提供新的方法。 方法:(1)用分散酶和胶原酶二步消化法分离人皮肤成纤维细胞,并进行原代培养。(2) MTT法检测维生素C、胰岛素和角质形成细胞培养上清液对成纤维细胞增殖的影响以及成纤维细胞培养上清液对角质形成细胞增殖的影响。(3)成纤维细胞长期体外培养使其形成纤维膜,并通过倒置显微镜和染色对纤维膜进行观察。 结果:(1)成纤维细胞接种24 h后,细胞逐渐呈放射状伸展,中心随之扁平,接种2~3 d细胞开始缓慢生长,约5~7 d汇合成片,HE染色和波形蛋白(vimentin)染色证明是成纤维细胞。(2)与对照组相比,低浓度的维生素C(25μg/ml、50μg/ml、75μg/ml)对体外培养的成纤维细胞有促增殖作用(P均0.05),且50μg/ml浓度组的促增殖作用大于25μg/ml浓度组(P0.05),而与75μg/ml浓度组相比无统计学差异(P0.05),但高浓度的维生素C(100μg/ml、125μg/ml)对成纤维细胞生长有明显的抑制作用(P 0.05),且后者的的抑制作用大于前者(P 0.05)。(3)胰岛素(2.5μg/ml、5μg/ml、7.5μg/ml和10μg/ml)促进成纤维细胞的增殖(P均0.05),且5μg/ml浓度组的促增殖作用大于2.5μg/ml浓度组(P0.05),而后三个浓度组之间差异无显著性(P0.05),但12.5μg/ml的胰岛素对成纤维细胞有明显抑制增殖作用(P0.05)。(4)角质形成细胞培养上清液对成纤维细胞的增殖有促进作用,且50%浓度组25%浓度组12.5%浓度组(P0.05),而50%浓度组与75%浓度组相比,差异无统计学意义(P0.05)。(5)成纤维细胞培养上清液能促进角质形成细胞的增殖,25%浓度组的促增殖作用大于12.5%浓度组(P 0.05),但25%、50%和75%各浓度组间比较,差异无统计学意义(P0.05)。(6)含胰岛素(2.5μg/ml,5μg/ml)或维生素C+胰岛素(50μg/ml的维生素C㧏5μg/ml的胰岛素)的培养基可促进体外培养的成纤维细胞形成纤维膜,且后者形成的纤维膜较前者更厚。(7) HE染色示纤维膜由浅红染色的细胞外基质组成;Masson-trichrome染色结果显示蓝色的胶原纤维。 结论:(1)用分散酶和胶原酶二步消化法对成纤维细胞进行原代分离、培养,经HE染色和免疫组化染色鉴定为成纤维细胞。(2)低浓度维生素C(25μg/ml~ 75μg/ml)和胰岛素(2.5μg/ml~10μg/ml)可促进成纤维细胞的生长,而高浓度维生素C(≥100μg/ml)和胰岛素(≥12.5μg/ml)则抑制成纤维细胞增殖,为纤维膜的培养提供了实验方法学依据。(3)角质形成细胞培养上清液可促进成纤维细胞生长,反之亦然。(4)胰岛素或胰岛素+维生素C能促进成纤维细胞体外形成类似于真皮的纤维膜,可用于人工真皮的构建。
[Abstract]:Objective: to study the effects of vitamin C and insulin on the proliferation of fibroblasts and the effects of vitamin C and insulin on fibroblast membrane formation in vitro.It provides a new method for the construction of dermis substitutes without xenogeneic collagen in the future.Methods Human skin fibroblasts were isolated by two-step digestion of dispersase and collagenase.The effects of vitamin C, insulin and keratinocyte culture supernatant on the proliferation of fibroblasts and the effects of fibroblast culture supernatants on the proliferation of keratinocytes were detected by MTT method.Cells were cultured in vitro for a long time to form fibrous membranes.The fiber membrane was observed by inverted microscope and staining.Results (1) after 24 h of inoculation, the fibroblasts gradually spread out radially, and the center of the fibroblasts gradually flattened, and the cells began to grow slowly on the 3rd day after inoculation.The results of HE staining and vimentin staining showed that the cells were fibroblasts.Low concentration of vitamin C 25 渭 g / ml 50 渭 g / ml 75 渭 g / ml) could promote proliferation of fibroblasts cultured in vitro (P < 0.05), and the effect of 50 渭 g/ml group was greater than that of 25 渭 g/ml group (P 0.05), but there was no significant difference compared with 75 渭 g/ml group (P 0.05), but high concentration of vitamin C 100 渭 g / ml ~ (125 渭 g 路ml ~ (-1) 路ml ~ (-1))The inhibitory effect of the latter on fibroblast growth was significantly higher than that on the former group (P 0.05), and the inhibitory effect of the latter was greater than that of the former group (2.5 渭 g / ml, 7.5 渭 g / ml and 10 渭 g / ml). The proliferation of fibroblasts was promoted by 5 渭 g/ml group (P < 0.05), and the effect of 5 渭 g/ml group was higher than that of 2.5 渭 g/ml concentration group (P 0.05), and the inhibitory effect of the latter was higher than that of the former group (P 0.05 渭 g / ml and 10 渭 g / ml), and the inhibitory effect of the latter was higher than that of the latter group.There was no significant difference between the latter three concentration groups, but 12.5 渭 g/ml insulin could significantly inhibit the proliferation of fibroblasts. The supernatant of keratinocyte culture could promote the proliferation of fibroblasts.And 50% concentration group 25% concentration group 12.5% concentration group P0.05N group, and 50% concentration group and 75% concentration group compared with 75% concentration group, and 50% concentration group compared with 75% concentration group.There was no significant difference in the effect of fibroblast culture supernatant on the proliferation of keratinocytes in the 25% concentration group, which was higher than that in the 12.5% concentration group (P 0.05), but there was a comparison between the 25% and 75% concentration groups.The medium containing 2.5 渭 g / ml insulin (5 渭 g / ml) or 50 渭 g/ml of vitamin C insulin (50 渭 g/ml) could promote fibroblasts to form fibroblasts in vitro.The fiber membrane formed by the latter was thicker than that of the former. (7) HE staining showed that the fibrous membrane was composed of light red stained extracellular matrix and Masson-trichrome staining showed blue collagen fibers.Conclusion the fibroblasts were isolated and cultured by two steps digestion of dispersase and collagenase. The fibroblasts were identified as fibroblasts by HE staining and immunohistochemical staining. Low concentration of vitamin C (25 渭 g / ml ~ 75 渭 g / ml) and insulin (2.5 渭 g/ml~10 渭 g / ml) could promote the growth of fibroblasts.However, high concentrations of vitamin C (鈮，
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