本文選題：角質(zhì)形成細(xì)胞 + 成纖維細(xì)胞�。� 參考：《蘇州大學(xué)》2010年碩士論文

【摘要】： 目的:通過研究角質(zhì)形成細(xì)胞、維生素C和胰島素對(duì)成纖維細(xì)胞增殖的影響以及維生素C和胰島素對(duì)成纖維細(xì)胞體外形成纖維膜的作用,為今后構(gòu)建不含異種膠原的真皮替代物提供新的方法。 方法:(1)用分散酶和膠原酶二步消化法分離人皮膚成纖維細(xì)胞,并進(jìn)行原代培養(yǎng)。(2) MTT法檢測(cè)維生素C、胰島素和角質(zhì)形成細(xì)胞培養(yǎng)上清液對(duì)成纖維細(xì)胞增殖的影響以及成纖維細(xì)胞培養(yǎng)上清液對(duì)角質(zhì)形成細(xì)胞增殖的影響。(3)成纖維細(xì)胞長期體外培養(yǎng)使其形成纖維膜,并通過倒置顯微鏡和染色對(duì)纖維膜進(jìn)行觀察。 結(jié)果:(1)成纖維細(xì)胞接種24 h后,細(xì)胞逐漸呈放射狀伸展,中心隨之扁平,接種2~3 d細(xì)胞開始緩慢生長,約5~7 d匯合成片,HE染色和波形蛋白(vimentin)染色證明是成纖維細(xì)胞。(2)與對(duì)照組相比,低濃度的維生素C(25μg/ml、50μg/ml、75μg/ml)對(duì)體外培養(yǎng)的成纖維細(xì)胞有促增殖作用(P均0.05),且50μg/ml濃度組的促增殖作用大于25μg/ml濃度組(P0.05),而與75μg/ml濃度組相比無統(tǒng)計(jì)學(xué)差異(P0.05),但高濃度的維生素C(100μg/ml、125μg/ml)對(duì)成纖維細(xì)胞生長有明顯的抑制作用(P 0.05),且后者的的抑制作用大于前者(P 0.05)。(3)胰島素(2.5μg/ml、5μg/ml、7.5μg/ml和10μg/ml)促進(jìn)成纖維細(xì)胞的增殖(P均0.05),且5μg/ml濃度組的促增殖作用大于2.5μg/ml濃度組(P0.05),而后三個(gè)濃度組之間差異無顯著性(P0.05),但12.5μg/ml的胰島素對(duì)成纖維細(xì)胞有明顯抑制增殖作用(P0.05)。(4)角質(zhì)形成細(xì)胞培養(yǎng)上清液對(duì)成纖維細(xì)胞的增殖有促進(jìn)作用,且50%濃度組25%濃度組12.5%濃度組(P0.05),而50%濃度組與75%濃度組相比,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。(5)成纖維細(xì)胞培養(yǎng)上清液能促進(jìn)角質(zhì)形成細(xì)胞的增殖,25%濃度組的促增殖作用大于12.5%濃度組(P 0.05),但25%、50%和75%各濃度組間比較,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。(6)含胰島素(2.5μg/ml,5μg/ml)或維生素C+胰島素(50μg/ml的維生素C㧏5μg/ml的胰島素)的培養(yǎng)基可促進(jìn)體外培養(yǎng)的成纖維細(xì)胞形成纖維膜,且后者形成的纖維膜較前者更厚。(7) HE染色示纖維膜由淺紅染色的細(xì)胞外基質(zhì)組成;Masson-trichrome染色結(jié)果顯示藍(lán)色的膠原纖維。 結(jié)論:(1)用分散酶和膠原酶二步消化法對(duì)成纖維細(xì)胞進(jìn)行原代分離、培養(yǎng),經(jīng)HE染色和免疫組化染色鑒定為成纖維細(xì)胞。(2)低濃度維生素C(25μg/ml~ 75μg/ml)和胰島素(2.5μg/ml~10μg/ml)可促進(jìn)成纖維細(xì)胞的生長,而高濃度維生素C(≥100μg/ml)和胰島素(≥12.5μg/ml)則抑制成纖維細(xì)胞增殖,為纖維膜的培養(yǎng)提供了實(shí)驗(yàn)方法學(xué)依據(jù)。(3)角質(zhì)形成細(xì)胞培養(yǎng)上清液可促進(jìn)成纖維細(xì)胞生長,反之亦然。(4)胰島素或胰島素+維生素C能促進(jìn)成纖維細(xì)胞體外形成類似于真皮的纖維膜,可用于人工真皮的構(gòu)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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