体内外构建毛囊的探索研究

发布时间：2018-06-24 17:02

本文选题：细胞培养 + 外根鞘细胞　；参考：《昆明医学院》2011年硕士论文

【摘要】：目的：分离和培养出毛囊来源的种子细胞,体外和体内诱导毛囊形成,并探讨毛囊的生物学特征和种子细胞在毛囊形成中的作用。方法：(1)用酶消化法结合组织块培养法从新生儿包皮获取皮肤成纤维细胞(Fb)；(2)购买人的毛囊外根鞘细胞株(HHFORSC)和人毛囊毛乳头细胞株(HHDPC),并行复苏、培养和传代；(3)将毛囊外根鞘细胞(ORSC)、毛乳头细胞(DPC)和丝裂霉素干预后的成纤维细胞按一定组合制成细胞悬液,并按不同的接种顺序接种于海藻酸钠3D细胞支架上,构建毛囊的体外三维模型,培养8周,行HE染色光镜观察毛囊形成的情况；同时将该三维模型移植入bal/bcl裸鼠皮下,饲养裸鼠8周,移植部位取材后分别行HE染色、免疫组化和电镜观察毛囊形成的情况。结果：(1)成功获得人Fb；(2)复苏、培养和传代的HHFORSC、HHDPC和自行分离、培养的Fb生长正常；(3)体外构建的毛囊三维模型,HE染色可见支架中有均匀散在分布的毛囊混合细胞,未见到角化物质及毛囊结构；(4)裸鼠皮下移植物HE染色可见细胞聚集成团,有呈环状排列的毛囊样结构,CK14,CK15、β1整合素和波形丝蛋白染色阳性；电镜下模型中可见贴附在支架上的毛囊细胞和红细胞。(5)先接种用丝裂霉素干预的成纤维细胞1×103于海藻酸钠3D细胞支架上培养一周后再接种DPC:ORSC (1:5)细胞悬液,裸鼠体内移植物HE染色不仅可见明显的毛囊样结构形成,而且形成毛囊样结构的数目较多；(6)MTT显示浓度为10ng/ml的骨形成蛋白4(BMP-4)对毛乳头细胞和外根鞘细胞均具有明显的促增值作用；(7)种子细胞的体外培养基用KGM组毛囊细胞聚集成细胞团块,部分呈环状排列,而用MSCM组则有细胞团样结构,未见环状结构。结论：本试验使用的酶消化法和组织块培养法获得的Fb保持了良好的生长特性；体外毛囊三维模型培养未能诱导出毛囊样结构,但在bal/bcl裸鼠体内诱导出了毛囊样结构,且该模型中DPC保持了诱导毛囊形成的能力,ORSC保持了毛囊干细胞的特点。此外,本试验探索出了诱导毛囊形成的最佳细胞组合、接种顺序和比例是先接种用丝裂霉素干预的成纤维细胞1×103于海藻酸钠3D细胞支架上,培养一周后再接种DPC:ORSC(1:5)细胞悬液；还证实了浓度为10ng/ml的BMP-4能明显促进DPC和ORSC的增殖；种子细胞的体外培养基用KGM明显优于MSCM。本试验为成功重建毛囊指明了方向。
[Abstract]:Aim: to isolate and culture the seed cells from hair follicles and induce hair follicle formation in vitro and in vivo, and to investigate the biological characteristics of hair follicles and the role of seed cells in hair follicle formation. Methods: (1) Human hair follicle outer root sheath cell line (HHFORSC) and human hair follicle hair papilla cell line (HHDPC) were obtained from the skin fibroblasts (FB); (2) of newborn by enzyme digestion and tissue mass culture, and were resuscitated, cultured and subcultured. (3) hair follicle outer root sheath cells (ORSC), dermal papilla cells (DPC) and fibroblasts treated with mitomycin were combined to make cell suspension and inoculated on sodium alginate 3D cell scaffold according to different inoculation order to construct the three-dimensional model of hair follicle in vitro. After 8 weeks of culture, the hair follicle formation was observed by HE staining, and the three dimensional model was transplanted into bal/bcl nude mice subcutaneously and fed for 8 weeks. The hair follicle formation was observed by HE staining, immunohistochemistry and electron microscope. Results: (1) Human Fb was successfully obtained; (2) HHFORSC HHDPC was resuscitated, cultured and isolated, and the cultured Fb grew normally; (3) the hair follicle 3D model was constructed in vitro and stained with HE, and there were hair follicle mixed cells scattered in the scaffold. No keratocystis and hair follicles were found. (4) the cells gathered into clusters in HE staining of subcutaneous grafts in nude mice, and the hair follicle-like structures, such as CK14, CK15, 尾 1 integrin and vimentin, were positive for the expression of CK14, CK15, 尾 1 integrin and vimentin, respectively. The hair follicle cells and erythrocytes attached to the scaffold were observed under electron microscope. (5) fibroblasts treated with mitomycin were first cultured on sodium alginate 3D scaffold for one week and then inoculated with DPC: ORSC (1:5) cell suspension. He staining of grafts in nude mice not only showed the formation of hair follicle-like structure, but also formed a large number of hair follicle-like structures. (6) Bone morphogenetic protein 4 (BMP-4) at the concentration of 10ng/ml significantly promoted the proliferation of both dermal papilla cells and outer root sheath cells. In MSCM group, there were cell clusters like structure, no ring structure. Conclusion: the Fb obtained by enzyme digestion and tissue mass culture has good growth characteristics, and hair follicle-like structure can not be induced by three-dimensional model culture of hair follicles in vitro. But hair follicle-like structure was induced in nude mice. In this model, the ability of bal/bcl to induce hair follicle formation was maintained. ORSC maintained the characteristics of hair follicle stem cells. In addition, the optimal cell combination for hair follicle formation was found in this experiment. The inoculation sequence and proportion were as follows: 1 脳 103 fibroblasts treated with mitomycin were first inoculated on sodium alginate 3D scaffold, and then inoculated with DPC: ORSC (1:5) cell suspension for one week. It was also confirmed that BMP-4 at the concentration of 10ng/ml could significantly promote the proliferation of 10ng/ml and the seed cell culture medium in vitro was better than that of 10ng/ml. This experiment pointed out the direction of successful hair follicle reconstruction.
【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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