【作者】 崔志峰；

【导师】 王慧；

【作者基本信息】 山东农业大学 ， 动物遗传育种与繁殖， 2013， 博士

【摘要】 以济宁青山羊为动物模型，从毛囊细胞体外分离培养和体外毛囊重建研究角度阐明毛囊的生长发育规律、影响因素及毛囊真皮与表皮细胞的互作机制，不仅可为提高山羊毛绒产量和品质的育种改良奠定基础；还可为加速提高毛绒品质、数量和揭示毛发再生机理乃至医学领域的人类毛发研究等提供借鉴。毛囊细胞体外分离培养技术的完善，体外重塑毛囊模型的建立，可为研究毛囊生长与分化机制，相关细胞因子对皮肤毛囊细胞损伤修复的作用机理，尤其是哺乳动物毛纤维形成机制提供良好模型，也是研究相关细胞因子以及激素对毛囊发育、羊毛品质和黑色素形成与调控机理的基础。本研究在成功构建了多种山羊皮肤毛囊细胞体外分离培养鉴定技术平台的基础上，优化了适宜的培养方法和培养体系，成功构建了毛囊体外重塑模型与毛纤维体外生成体系，可为进一步深入研究毛囊分化与发育的影响因素及其所涉及到的一系列生理现象，如皮肤毛囊细胞分化、真皮和表皮细胞间的互作、皮肤毛囊损伤和被清理机制等研究奠定基础。主要研究内容、方法和结果如下：一、山羊毛囊外根鞘细胞体外培养体系的优化无菌分离活体青山羊羔羊的次级毛囊，采用机械分离与酶消化相结合的方法，分离纯一的毛囊外根鞘细胞（ORSC），培养于含有表皮生长因子（EGF）、类胰岛素生长因子-I（IGF-I）和氢化可的松（hydrocortisone）的无血清角质细胞培养液中，置5%CO2浓度的37℃培养箱中启动原代培养。待原代细胞长成良好的单层后即可进行传代培养，细胞经传代培养至8~10代时，更换含EGF、IGF-I、氢化可的松和2%FBS的DMEM/F12培养液进行长期培养，更换培养液后可见细胞生长状态逐渐趋于稳定。选取稳定传代的ORSC进行生物学特性研究与鉴定。结果表明：该细胞的群体倍增时间为51.9h；培养细胞的染色体数仍以2n=60为主，细胞免疫化学鉴定结果表明，外根鞘细胞角蛋白19表达呈阳性，证实本实验分离培养的细胞确为由毛囊干细胞分化来的外根鞘细胞。二、山羊毛乳头细胞的分离培养与鉴定将中性蛋白酶与胶原酶消化及预铺设促贴壁培养基质相结合，分离并纯化山羊DPC，培养于含有表皮生长因子（EGF）、碱性成纤维细胞生长因子（bFGF）和10%FBS的DMEM/F12培养液中，放入37℃CO2培养箱中进行原代培养。待原代细胞长至完全汇合并进行传代培养后，更换含EGF、bFGF和2%FBS的MEM与DMEM/F12（1:1）复合培养液继续传代培养，更换培养液后可见细胞生长状态逐渐趋于稳定。选取稳定传至第7代的DPC进行生物学特性研究与鉴定。结果表明：该细胞的平均克隆形成率为60%，细胞增殖活力旺盛；培养细胞的染色体数仍以2n=60为主，细胞免疫荧光鉴定结果表明，体外培养的DPC α-SMA和Vimentin表达均呈阳性，充分证实本实验分离培养的细胞确为毛囊真皮源性的毛乳头细胞。三、山羊真皮成纤维细胞的体外培养及其细胞系的建立利用中性蛋白酶（dispase）4℃冷消化法分离皮肤的表皮层和真皮层，以利于纯化培养真皮成纤维细胞。然后，采用原代外植与酶消化相结合的方法，分离纯化的DFB：即先利用Dispase酶分离皮肤表、真皮层后，剪切真皮部分成组织小块，再对小组织块进行酶（胰蛋白酶）消化处理，随即将真皮外植块接种于6孔培养板底壁上。待贴牢后添加含有碱性成纤维细胞生长因子（basic fibroblast growth factor，bFGF）、青霉素、链霉素、泰乐菌素（tylosin）和10%FBS的DMEM/F12培养液中，置5%CO2浓度的37℃饱和湿度培养箱中启动原代培养。当培养板中的真皮外植块迁移出大量细胞时（约5~6天），换为含有bFGF、青霉素、链霉素、泰乐菌素（tylosin）和5%FBS的DMEM培养液继续进行培养，每3天更换1次培养液。待原代真皮成纤维细胞生长至汇合时，进行传代，并于第一次传代时更换为25cm2培养瓶中培养，以利于真皮成纤维细胞在体外长期传代培养与冻存。本研究所建立的DFB细胞系已经稳定传至56代以上，其生长分裂状态良好，细胞遗传性状稳定。四、山羊体外重塑毛囊模型的建立和维持在无菌冰浴条件下，将鼠尾胶原、透明质酸、硫酸软骨素A按比例依次混合到MEM与DMEM/F12（1:1）复合培养液中，制成凝胶。调节pH后加入24孔板中。收获前期分离培养与鉴定的纯化毛乳头、真皮成纤维细胞，用含EGF、bFGF和5%FBS的MEM与DMEM/F12（1:1）混合培养液制成细胞混悬液，用无菌吸管将细胞混悬液加入24孔板中，待其在常温下形成真皮细胞胶原凝胶后，置于37℃、5%CO2的饱和湿度环境中进行培养。每天在显微镜下观察DPC与DFB的生长增殖情况。待毛囊真皮细胞达到完全汇合后，表面覆盖预先制备的ORSC细胞混悬液，继续培养3~5天，观察ORSC于毛囊真皮细胞胶原凝胶上形成完整单层后，去除旧的培养液，加入少量（恰好覆盖表面ORSC细胞即可）含IGF-I、EGF的无血清角质细胞培养液，在37℃、5%CO2的饱和湿度环境中进行气-液界面培养，每两天更换一次表层培养基。经显微观察拍摄、测微尺测量与实时荧光定量PCR检测，结果显示，在进行气液界面培养初期，毛乳头细胞即呈现凝集性生长趋势，而表面的毛囊外根鞘细胞亦呈集落样散在分布于真皮成纤维细胞层中。随着气液界面培养的进行，毛乳头细胞进一步凝集性生长，部分外根鞘细胞集落通过凝胶中的真皮成纤维细胞单层迁移聚集在毛乳头细胞团周围，并不断包绕，分化形成圆球样的类毛球结构贴附在真皮成纤维细胞层上。约经过10天的体外培养，由几种毛囊细胞形成的圆球状结构逐渐向外围拉伸扩张，内部的毛乳头细胞向一端迁移生长，最终由毛球样结构伸展延长成类毛囊结构，并有毛纤维从基部长出，部分生长出的毛纤维颜色为浓黑色，内含黑色素。各检测数据显示，体外重建的毛囊结构与体内正常毛囊比较无论在组织结构、毛纤维生长速度，还是毛囊发育关键基因的表达调控方面，均差异不显著。本研究优化并建立了山羊毛囊细胞体外培养条件和体系，研究了细胞因子对毛囊真皮表皮细胞的影响，成功建立了山羊毛囊体外诱导重建体系，为揭示山羊毛囊分化发育与羊毛生长的影响因素和调控机理奠定了基础和提供了模型；并为医学研究和发现毛发疾患病因、探讨药物治疗作用机理等提供了借鉴。本研究所涉及的细胞因子和培养条件等，多数反映了一般实验条件下这些常见因素的互作，期望能为人类毛发再生和脱发问题研究提供借鉴。更多还原

【Abstract】 Taking Jining Grey goats as animal models，clarifies the growth regular of hair follicle,impact factors and the interaction mechanism of hair follicle dermal cells and epidermal cellsfrom isolation and culture of the hair follicle cells and reconstitution of hair follicle in vitro.Not only to lay the foundation for improving the quality of the breeding improved ofCashmere quality, but also to offer reference for improving the wool quality, quantity, qualityof fur pattern and human hair research in medical area. Improvement technique of isolatingand culturing the hair follicle cells, constructing reconstitution model of hair follicles in vitrocan lay the foundation for further study in hair follicle differentiation and development,mechanism of cell factors on hair follicle cell injury and skin cell repair, mechanism of thecell factors and hormone on follicle development, wool quality and melanin formation andregulation. The present study based on the successful construction of the platform oninsolation, culture and identification of a variety of skin cells and hair follicle cells from goatin vitro, screening the optimum culture methods and system and building cell line impactfactors and involved many physiological phenomena, such as skin and hair follicles celldifferentiation, interaction between dermal and epidermal cell, mechanism on skin and hairfollicle damage and cleaning.I. Improvement of outer root sheath cells culture system from goat hair follicle in vitro.Adopting enzyme digestion in conjunction with mechanical methods sterile isolatesentire hair follicle, the purified ORS cells were cultured in serum-free keratinocyte mediumcontaining epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I) andhydrocortisone. The cells were incubated in a5%CO2atmosphere at37℃in the start ofprimary culture. The ORS cells were split to subcultures when the primary cells formed goodmonolayer. ORS cells were subcultured to8th to10th passages, the medium was replacedwith DMEM/F12medium containing EGF, IGF-I, hydrocortisone and2%FBS for the longterm culture. The cells showed gradually stabilized growth after changing the medium. Detected and identified the biological characteristic of ORS cells. The results show that thecultured cells population doubling time was51.9h, chromosome number is still dominated by2n=60. The results of immunocytochemical staining showed that the cytokeratin19expression was positive, proved that the cultured cells is outer root sheath cells from hairfollicle stem cell differentiation.II. Isolation and identification of dermal papilla cells from goat.The dispase combined with collagenase digestion and laying of adherent culture matrix,separation and purification of DP cells, cultured in DMEM/F12medium containing epidermalgrowth factor (EGF), basic fibroblast growth factor (bFGF) and10%FBS in primary culture.When the primary cells were completely confluent subcultured, the medium was replacedwith MEM and DMEM/F12with EGF, bFGF and2%FBS (1:1) composite medium tocontinue to subculture. The cells showed gradually stabilized growth after changing themedium. The DP cells at7th passage were selected to study on biological characteristics andidentification. The results show that the average cloning efficiency of cell is60%, suggest thecells proliferation vitality were luxuriant. The results of chromosomal analysis indicated thatmajority of the cells were2n=60. The results of immune-fluorescence showed that theexpression of α-SMA and Vimentin were positive in DPC and fully confirmed the subculturedcells. The results proved that the dermal papilla cells were hair follicle dermal source.III. Culture of dermal fibroblasts from the goat and establishment of the dermalfibroblast cell line.The method of4℃dispase cold digestion was used to separate the skin epidermis anddermis, in order to culture the purification of dermal fibroblasts. Then, adopting the primaryexplant combined with enzyme digestion separated and purified DFB. At first dispase wasused to separate epidermis and dermis from skin, and the sterile dermal part was gentlyminced into small pieces. The small tissue pieces were enzyme by trypsin, and then dermalexplants were seeded on the bottom of6-well plates. DMEM/F12medium with containingbasic fibroblast growth factor (bFGF), penicillin, streptomycin, tylosin and10%FBS wasadded to each well of the6-well plates after the pieces attached firmly. The cells wereincubated in a5%CO2atmosphere at37℃in the start of primary culture. When a largenumber of cells were migrated out from cultured dermal explant (about5~6days), the medium was replaced with DMEM medium containing bFGF, penicillin, streptomycin,tylosin and5%FBS to subculture, the medium was replace every3days. The cells werereplaced to the25cm2flasks at the first passage time when the primary dermal fibroblastswere grown to confluency, in favor of dermal fibroblasts cultured long term subculture andcryopreservation in vitro. The stable DFB cell line was established in this research hassubcultured to the56passages, and the growth and proliferation of cells were in goodcondition, genetic stability.IV. Establish and maintain the reconstitution model of hair follicle in vitro.In the sterile ice bath, the rat tail collagen, hyaluronic acid and chondroitin sulfate A weremixed proportion to MEM and DMEM/F12(1:1) composite medium to make of gel, and thenadded to24-well plates after regulated pH of the gel. The purified dermal papilla cells anddermal fibroblasts that were isolated and identificated the early stage were harvest, the cellswere suspended by MEM and DMEM/F12containing5%FBS (1:1) composite medium.Sterile cell suspension was added in24-well plates, formed the dermal cell collagen gel. Thecell collagen gel was incubated in37℃and5%CO2humidity environmental conditions. Thedaily growth and proliferation of DPC and DFB were observed under microscope. When thehair follicle dermal cells reached completely confluence, prepared ORS cell suspension addedthe surface, cultured for3~5days. Observed ORS cells in the hair follicle dermal cell collagengel to form a complete monolayer, removed the old medium and added a small amountK-SFM (just covered the surface of ORS cells) containing IGF-I, EGF at37℃,5%CO2saturated humidity of gas-liquid interface culture, replaced the surface medium every twodays. Through microscopic observation, micrometer measurement and real-time fluorescencequantitative PCR detection, the results show that dermal papilla cells present agglutinativegrowth trend in the gas-liquid interface culture early, but the hair follicle outer root sheathcells and scattered colonies in the dermal fibroblast layer. With the gas-liquid interface culture,dermal papilla cells further agglutinative growth, part of the outer root sheath cells colony bygel dermal fibroblast monolayer migration gathered around the dermal papilla cells, andconstantly surrounded, differentiate into ball kind of type of hair ball structure attached to thedermal fibroblast cell layer. Spherical structure formed by several hair follicle cells graduallyto peripheral tension expansion after10days of culture in vitro, dermal papilla cells to end internal migration growth, and ultimately by the hair bulb structure extensional extend intothe class structure of hair follicle, and hairy fibers from the radical minister, wool fiber colorpart of the students to grow as thick black, with black pigment. The testing data shows, invitro reconstruction of hair follicle structure and normal hair follicles are both in theorganizational structure, wool growth rate, the key gene and hair follicle developmentregulation of expression, there is no significant differences.This study established and optimized conditions and culture system of hair follicle cellsin vitro from goat. Studied the effects of impact factors on hair follicle dermal cells andepidermal cells, successfully established a reconstruction induction system of hair follicle invitro from goat. This provide animal models and lay the foundation for people to reveal theand regulatory mechanism of differentiation and development of goat hair follicle and woolgrowth; provides a reference for medical research and discovery cause of hair disorders, themechanism of such drug treatment. Related factors involved in this study and the cultureconditions reflect the most common interactions among these factors in the experimentalconditions, provide reference for human hair regeneration and canities research.更多还原