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  本文選題：成纖維細胞　切入點：表皮細胞　出處：《山東大學》2011年碩士論文

【摘要】：目的日常生活中,大面積深度燒傷、糖尿病、創(chuàng)傷等因素常造成正常皮膚結構完整性及生理功能的破壞。如何有效的修復創(chuàng)面、重建皮膚正常組織結構恢復皮膚正常生理功能,成為目前實驗研究以及臨床治療的難點。皮膚損傷的修復是-個非常復雜的過程,由多種生長因子及細胞因子介導,其中角質細胞生長因子(KGF)發(fā)揮了重要的作用。角質細胞生長因子為成纖維細胞生長因子家族的一員,它通過與受體(KGFR)結合發(fā)揮作用,是一種促上皮細胞增殖的生長因子,與創(chuàng)面愈合息息相關,還與免疫重建、器官發(fā)育、腫瘤發(fā)生等多方面關系密切。該因子由間質細胞分泌,其表達受IL-1、血小板衍生生長因子BB、轉化生長因子、腫瘤壞死因子等細胞因子的正調控作用影響,通過間充質-上皮相互作用方式,對多種組織器官,包括肺、皮膚、呼吸道、頭發(fā)毛囊、胃腸道、膀胱等的上皮損傷的修復發(fā)揮重要作用,但是KGF的生物學性質尚未完全了解。本實驗旨在研究成纖維細胞產(chǎn)生的角質細胞生長因子(KGF)對表皮細胞增殖的影響。 方法取16-30歲患者手術切除的包皮修剪至全厚皮片,外分離培養(yǎng)成纖維細胞和表皮細胞,取第二代的成纖維細胞提取總RNA采用逆轉錄-聚合酶鏈反應法(RT-PCR)檢測人皮膚成纖維細胞中KGF基因的轉錄水平,對第2代的成纖維細胞培養(yǎng)48 h后,收集細胞培養(yǎng)液用酶聯(lián)免疫吸附法(ELISA)檢測培養(yǎng)液中KGF的濃度,之后對第2代表皮細胞進行傳代,分別用K-SFM(不含KGF),K-SFM(含1ng/mL的KGF), DMEM(含10%的FBS)和成纖維細胞培養(yǎng)液培養(yǎng)24h、36h、48h、60h、72h,用細胞計數(shù)法、MTT法和免疫熒光的方法檢測各實驗組培養(yǎng)液對表皮細胞增殖的促進作用。最后對所得數(shù)據(jù)經(jīng)行統(tǒng)計學處理。 結果RT-PCR結果顯示,可擴增獲得KGF,說明人皮膚成纖維細胞中KGF轉錄水平顯著,酶聯(lián)免疫吸附法(ELISA)可檢測到人皮膚成纖維細胞培養(yǎng)液中存在較高水平的KGF,細胞計數(shù)結果和MTT結果表明,添加KGF的K-SFM和成纖維細胞培養(yǎng)液均能夠促進表皮細胞增殖,亦可保持表皮細胞的活性,并用免疫熒光檢測方法得出,人皮膚成纖維細胞培養(yǎng)液中的KGF能夠與表皮細胞表面的KGF特異性受體相結合。 結論本實驗得出體外分離培養(yǎng)的人皮膚成纖維細胞中KGF轉錄水平顯著,成纖維細胞培養(yǎng)液中較其他對照組能夠檢測到高濃度的KGF,且各組差異具有統(tǒng)計學意義(P0.01),說明成纖維細胞能夠合成和釋放KGF,且可與表皮細胞表面的KGF特異性受體相結合而發(fā)揮其特異性作用,成為其促進表皮細胞增殖的重要因素,從而促進創(chuàng)面修復的進程。
[Abstract]:Objective in daily life, extensive deep burns, diabetes mellitus, trauma and other factors often cause damage to normal skin structure integrity and physiological function.How to effectively repair the wound and reconstruct the normal tissue structure of the skin to restore the normal physiological function of the skin has become a difficult point in the current experimental research and clinical treatment.Skin injury repair is a very complex process, mediated by a variety of growth factors and cytokines, including keratinocyte growth factor (KGF) plays an important role.Keratinocyte growth factor is a member of the fibroblast growth factor family. It acts as a growth factor that promotes the proliferation of epithelial cells by binding to the receptor KGFR. It is closely related to wound healing, as well as to immune reconstruction and organ development.Tumorigenesis and other aspects are closely related.It is secreted by interstitial cells, and its expression is affected by the positive regulation of cytokines such as IL-1, platelet-derived growth factor (BBF), transforming growth factor (TGF-), tumor necrosis factor (TNF- 偽) and so on.The repair of epithelial injuries including lung, skin, respiratory tract, hair follicle, gastrointestinal tract, bladder and so on plays an important role, but the biological properties of KGF are not fully understood.The aim of this study was to investigate the effect of keratinocyte growth factor (KGF) produced by fibroblasts on the proliferation of epidermal cells.Methods the prepuce removed from patients aged 16-30 years was trimmed to full thickness skin grafts. Fibroblasts and epidermal cells were isolated and cultured in vitro.The total RNA extracted from the second passage fibroblasts was used to detect the transcription level of KGF gene in human skin fibroblasts by reverse transcription-polymerase chain reaction (RT-PCR). The second passage fibroblasts were cultured for 48 h.The concentration of KGF in the culture medium was detected by enzyme linked immunosorbent assay (Elisa), and then the second passage of epidermal cells was carried out.K-SFM (without 1ng/mL), DMEM (containing 10% FBS) and fibroblast culture medium were cultured for 24 h ~ 36 h ~ 48 h ~ 60 h ~ 72 h, respectively. The effects of culture medium on the proliferation of epidermal cells in each experimental group were detected by cell count method and immunofluorescence method.Finally, the data are statistically processed.Results the results of RT-PCR showed that KGF could be amplified, which indicated that the transcription level of KGF in human skin fibroblasts was significant.High level of KGF was detected by Elisa in human skin fibroblast culture medium. The results of cell count and MTT showed that both K-SFM and fibroblast supplemented with KGF could promote the proliferation of epidermal cells.The KGF in the culture medium of human skin fibroblasts could bind to the KGF specific receptor on the surface of epidermal cells.Conclusion the KGF transcription level in human skin fibroblasts isolated in vitro was significant.The high concentration of KGF was detected in fibroblast culture medium compared with other control groups, and the difference was statistically significant (P0.01), which indicated that fibroblasts could synthesize and release KGFs and be similar to the specific KGF receptor on the surface of epidermal cells.Combine to play its specific role,It is an important factor to promote the proliferation of epidermal cells, thus promoting the process of wound repair.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R363

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