毛囊真皮鞘细胞在皮肤创伤愈合中作用的研究

发布时间：2018-03-09 07:51

本文选题：毛囊　切入点：真皮鞘细胞　出处：《中国人民解放军军事医学科学院》2007年博士论文　论文类型：学位论文

【摘要】： [目的]如何促进创面愈合和减少瘢痕形成是创伤研究领域的重点问题之一。皮肤愈合与局部毛囊数量密切相关的有趣现象引起了关注。现已证实毛囊真皮鞘细胞具有成体干细胞特性。那么，它在创伤愈合中扮演怎样的角色?本课题对毛囊真皮鞘细胞在皮肤创伤愈合中的作用进行了较系统的研究，旨在为皮肤创/烧伤和病理性瘢痕治疗研究提供新的思路。 [方法]利用组织块法原代培养大鼠触须毛囊真皮鞘细胞，经免疫细胞化学和免疫荧光染色进行细胞鉴定，MTT检测和光镜、电镜观察细胞生长特性和超微结构特征；对BrdU细胞标记方法、检测手段和创面细胞移植时机进行优化，建立动物实验模型；通过超微结构扫描电镜定性观察和瘢痕三维重建体积定量建立瘢痕评价体系；大鼠背部创面同种毛囊真皮鞘细胞移植后，通过病理形态观察和图像分析对创面愈合及瘢痕形成进行动态定性和定量评价；采用RT-PCR、免疫细胞化学、免疫荧光和免疫印迹技术分别从基因或蛋白水平检测部分愈合相关蛋白在真皮鞘细胞中的表达。 [结果] (1)毛囊真皮鞘细胞具有的生长特性和结构特征：原代生长迟缓、传代后增殖旺盛，呈克隆样丛状生长；细胞呈纺锤形，，胞体丰满；核/浆比较大，核仁较多，胞膜下存在多量条带状致密斑。(2)建立了大鼠背部创面可示踪细胞移植模型：以终浓度为10μmol/L的BrdU标记细胞，于致伤前移植，术后14d的愈合组织中仍可见到植入细胞。(3)建立了皮肤创伤愈合瘢痕评价体系：以瘢痕中胶原纤维和胶原原纤维的排列作为定性指标；瘢痕体积作为定量指标。(4)毛囊真皮鞘细胞具有促进皮肤创面愈合的作用：术后各时间点细胞移植组创面收缩率均大于对照组(P＜0.01)；术后7d移植组新生肉芽组织中见不均匀分布的标记细胞，14d标记细胞减少并出现凋亡；移植组愈合过程中肉芽组织形成、表皮再生、胶原增生、瘢痕形成和组织改建均较对照组提前，所形成的瘢痕量呈减少趋势。(5)真皮鞘细胞的促愈机制：细胞中Ⅰ型前胶原蛋白mRNA高表达，bFGF、EGFR和TIMP1在蛋白水平较皮肤成纤维细胞高表达。 [结论]毛囊真皮鞘细胞为幼稚的间充质细胞，具有潜在增殖能力；在皮肤创伤修复过程中参与肉芽组织的形成和胶原合成，具有一定的加速创面收缩、促进愈合进程和减少瘢痕形成的作用；该作用与真皮鞘细胞某些愈合相关蛋白高表达有关。揭示了毛囊真皮鞘细胞的生物学特性，为其在创伤治疗中的应用提供了实验依据。
[Abstract]:[objective] how to promote wound healing and reduce scar formation is one of the key problems in the field of trauma research. The interesting phenomenon that skin healing is closely related to the number of local hair follicles has attracted attention. It has been proved that the dermal sheath of hair follicles is fine. Cells have the properties of adult stem cells. What role does it play in wound healing? The role of dermal sheath cells of hair follicles in the healing of skin trauma was studied systematically in order to provide new ideas for the treatment of skin trauma / burn and pathological scar. [methods] the primary cultured rat hair follicle dermal sheath cells were cultured by tissue block method. The cells were identified by MTT assay and light microscopy by immunocytochemistry and immunofluorescence staining. The growth and ultrastructural characteristics of the cells were observed by electron microscope. The BrdU cell labeling method, detection method and the time of wound cell transplantation were optimized to establish animal experimental model, and the scar evaluation system was established by qualitative observation of ultrastructural scanning electron microscope and 3D reconstruction volume of scar. The wound healing and scar formation were evaluated qualitatively and quantitatively by histopathological observation and image analysis after transplantation of dermal sheath cells of hair follicle of rat back wound, using RT-PCR, immunocytochemistry, immunocytochemistry, immunocytochemistry, immunocytochemistry, immunocytochemistry, immunocytochemistry, Immunofluorescence and Western blotting were used to detect the expression of partial healing related proteins in dermal sheath cells. [results] the growth characteristics and structural characteristics of hair follicle dermis sheath cells were as follows: primary growth retardation, vigorous proliferation after passage, clone like plexiform growth, spindle-shaped cells with full cell body, large nucleus / plasma and more nucleoli. The model of traceable cells transplantation in rat back wounds was established by using the BrdU labeled cells with a final concentration of 10 渭 mol/L before injury. Implantation cells were still found in the healing tissue 14 days after operation. The evaluation system of scar healing was established. The arrangement of collagen fibers and procollagen fibers in scar was used as qualitative index. Scar volume as a quantitative index. (4) hair follicle dermal sheath cells can promote the healing of skin wounds: at all time points after operation, the wound contraction rate in the cell transplantation group was higher than that in the control group (P < 0.01), but it was not found in the granulation tissue of the transplantation group on the 7th day after operation. The uniform distribution of labeled cells decreased and apoptosis appeared on the 14th day. Granulation tissue formation, epidermal regeneration, collagen proliferation, scar formation and tissue remodeling in the transplantation group were earlier than those in the control group. The healing mechanism of dermal sheath cells was as follows: type I procollagen mRNA was highly expressed in the cells and the expression of bFGFEGFR and TIMP1 was higher than that of skin fibroblasts at the protein level. [conclusion] hair follicle dermal sheath cells are immature mesenchymal cells with potential proliferative ability, and participate in the formation of granulation tissue and collagen synthesis in the process of skin wound repair. The role of promoting healing process and reducing scar formation is related to the overexpression of some healing related proteins in dermal sheath cells. The biological characteristics of dermal sheath cells in hair follicles are revealed and the experimental basis for its application in wound therapy is provided.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R363

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