KGF在成纤维细胞促进表皮细胞增殖作用中的研究

发布时间：2018-04-02 18:53

本文选题：成纤维细胞　切入点：表皮细胞　出处：《山东大学》2011年硕士论文

【摘要】：目的日常生活中,大面积深度烧伤、糖尿病、创伤等因素常造成正常皮肤结构完整性及生理功能的破坏。如何有效的修复创面、重建皮肤正常组织结构恢复皮肤正常生理功能,成为目前实验研究以及临床治疗的难点。皮肤损伤的修复是-个非常复杂的过程,由多种生长因子及细胞因子介导,其中角质细胞生长因子(KGF)发挥了重要的作用。角质细胞生长因子为成纤维细胞生长因子家族的一员,它通过与受体(KGFR)结合发挥作用,是一种促上皮细胞增殖的生长因子,与创面愈合息息相关,还与免疫重建、器官发育、肿瘤发生等多方面关系密切。该因子由间质细胞分泌,其表达受IL-1、血小板衍生生长因子BB、转化生长因子、肿瘤坏死因子等细胞因子的正调控作用影响,通过间充质-上皮相互作用方式,对多种组织器官,包括肺、皮肤、呼吸道、头发毛囊、胃肠道、膀胱等的上皮损伤的修复发挥重要作用,但是KGF的生物学性质尚未完全了解。本实验旨在研究成纤维细胞产生的角质细胞生长因子(KGF)对表皮细胞增殖的影响。方法取16-30岁患者手术切除的包皮修剪至全厚皮片,外分离培养成纤维细胞和表皮细胞,取第二代的成纤维细胞提取总RNA采用逆转录-聚合酶链反应法(RT-PCR)检测人皮肤成纤维细胞中KGF基因的转录水平,对第2代的成纤维细胞培养48 h后,收集细胞培养液用酶联免疫吸附法(ELISA)检测培养液中KGF的浓度,之后对第2代表皮细胞进行传代,分别用K-SFM(不含KGF),K-SFM(含1ng/mL的KGF), DMEM(含10%的FBS)和成纤维细胞培养液培养24h、36h、48h、60h、72h,用细胞计数法、MTT法和免疫荧光的方法检测各实验组培养液对表皮细胞增殖的促进作用。最后对所得数据经行统计学处理。结果RT-PCR结果显示,可扩增获得KGF,说明人皮肤成纤维细胞中KGF转录水平显著,酶联免疫吸附法(ELISA)可检测到人皮肤成纤维细胞培养液中存在较高水平的KGF,细胞计数结果和MTT结果表明,添加KGF的K-SFM和成纤维细胞培养液均能够促进表皮细胞增殖,亦可保持表皮细胞的活性,并用免疫荧光检测方法得出,人皮肤成纤维细胞培养液中的KGF能够与表皮细胞表面的KGF特异性受体相结合。结论本实验得出体外分离培养的人皮肤成纤维细胞中KGF转录水平显著,成纤维细胞培养液中较其他对照组能够检测到高浓度的KGF,且各组差异具有统计学意义(P0.01),说明成纤维细胞能够合成和释放KGF,且可与表皮细胞表面的KGF特异性受体相结合而发挥其特异性作用,成为其促进表皮细胞增殖的重要因素,从而促进创面修复的进程。
[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- 伪) 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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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