【作者】 谭挺；

【导师】 胡志奇；

【作者基本信息】 南方医科大学 ， 整形外科学， 2008， 硕士

【摘要】 背景:现代毛发修复外科技术,虽然其疗效肯定、持久,曾得到广大秃发患者的认可,但实践证明无论是哪一种修复技术的采用,都是以患者自体拥有充足的供区毛发为前提。对于秃区面积大,甚至毛发完全缺失的患者,这些修复技术常无法开展;即便开展,由于供区不足、移植成活率不稳定等因素,手术后的毛发密度也无法达到满意效果。况且任何外科手术都不会产生新的毛发,也不会增加毛发的数量。许多秃发患者毛发的丢失是进行性的,毛发修复也不能逆转或停止其丢失,更无法预测其发展情况。所以,供区毛发资源明显不足,成为现今毛发修复外科较难解决的问题,也对毛囊的组织工程技术快速发展提出了要求。毛囊是上皮组织中最复杂的器官,横跨表皮和真皮,不但其结构生理方面有很多特殊之处,而且有一套独特的免疫方式。毛囊是上皮与间充质相互作用的结果,由分别来源于中外两个胚层的上皮细胞和真皮细胞构成。毛囊的形态发生是细胞依附于细胞外基质在毛囊相关信号的诱导下定向移动与分化的结果,毛囊干细胞（HFSCs）对毛囊的形态发生和毛发的周期性生长起主导作用。因此建立HFSCs体外培养机制,对于详尽地了解HFSCs体外培养生长特点,以及如何诱导毛囊的发生,具有重要的意义。另一方面是干细胞研究必须要面对的干细胞纯化问题,目前普遍认为HFSCs定位于毛囊上段的隆突（Bulge）区,与基质具有高黏附性是HFSCs分离培养困难的重要因素之一。但目前传统的分离方法,操作繁杂、劳动量大,同时获得的毛囊隆突区细胞（HFBCs）中HFSCs纯度不高,易污染,因而不便于实验开展。目的:1.证实HFBCs在毛干上的定位,确定分离培养HFBCs的方法,并寻找技术改良。2.证实HFSCs的表面标记并尝试寻找纯化HFSCs的方法。3.进一步利用纯化后的HFSCs和毛乳头细胞（DPCs）混合进行体内诱导试验,尝试诱导出新生毛囊及毛发纤维。方法:1.鼠和人毛囊的显微解剖。采用显微解剖法分离出鼠及人的完整毛囊。将其置于倒置显微镜下观察其形态结构和区别。2.鼠HFBCs的分离、培养。显微分离培养法:无菌条件下取7日龄Wistar大鼠触须部皮肤,在含双抗（青链霉素）的D-Hanks’液中清洗2遍,在体视显微镜下用27号针头将毛囊从组织中分离出来,置于0.25%Dispase酶中,4℃消化2h后用D-Hanks’液清洗,在体视显微镜下轻轻挤压毛球部即可将毛囊Bulge区及毛干完整地分离出来,切去皮脂腺及毛囊中下段,保留Bulge区毛囊,将其置于含10%（体积比）胎牛血清的DMEM/F12补充培养基中进行培养。消化法:将组织法获得的毛囊Bulge区置于含0.25%Dispase酶和0.1%胶原酶的混合消化液中,37℃消化20min,吹打制悬,吸取悬液至离心管中并加入含血清培养基终止消化,剩余组织块继续加入消化液消化,并分次采集细胞悬液,直至组织块消化完全。收集离心后得到的细胞,并加入含血清培养基再次离心,最后加入含10%（体积比）胎牛血清的DMEM/F12补充培养基制成单细胞悬液,接种到培养瓶中。观察并比较两种方法获得的鼠HFBCs在DMEM/F12补充培养基中体外培养的形态学和生物学特点。3.人HFBCs的分离、培养。取头皮标本,参照显微分离培养法并加以改进,备皮后于70%乙醇中清洗,尽量除去皮下脂肪。将皮片切成长条,置入0.25%Dispase酶37℃消化2h。从真皮-皮下组织交界处横断头皮,用镊子从皮下组织端拉出毛囊,收集形态完好且处于生长期的毛囊,体视显微镜下分别在球部上端、皮脂腺下端横切毛囊,取中间部分,含双抗PBS漂洗3次,放入25mL培养瓶中,加入DMEM/F12补充培养基于37℃、5%CO₂孵箱中培养,每3d换液,观察细胞增殖能力及形态学特征。4.人HFSCs的鉴定与纯化。取少量HFBCs置于培养皿中,待其48小时重新贴壁后,经PBS漂洗3次（每次5min）,预冷丙酮固定10min,然后常规免疫细胞化学方法染色,检测K19的表达情况,以PBS代替一抗作阴性对照。将HFBCs重悬,调整为1×10⁸/mL,按1.0μg/mL浓度,加入FITC标记的鼠抗人CD200单抗,室温孵育30min。离心弃上清中未结合的抗体。加入1mL磁珠分选缓冲液重悬细胞,离心弃上清,PBS缓冲液清洗2次。取重悬后HFBCs与50μg免疫磁珠混合,室温反应30min,磁珠分离器分离8min,去除未与磁珠结合的细胞。将结合了靶细胞的免疫磁珠用1mL磁珠分选缓冲液洗5次。37℃、5%CO₂孵箱培养48h,使细胞与磁珠分离,得到较纯的HFSCs。5.人HFSCs的检测。细胞活性检测:将纯化后细胞悬液与0.4%苔盼蓝溶液按1:1体积比混合后,滴入细胞计数板置于显微镜下观察。细胞免疫荧光检测:分别于纯化前后取5μL细胞悬液于荧光显微镜下观察其表达CD200情况。HFSCs纯度及回收率分析:取CD200孵育后行磁珠分选前后的HFBCs进行实验,以对照试剂室温孵育30min的HFBCs为对照。各取5×10⁵个细胞上机。以对照管作为空白标定,记录标本的CD200阳性细胞百分率。流式数据采用Cellquest pro分析。纯度=阳性细胞数/细胞总数;回收率=（纯化后细胞总数×纯化后阳性细胞百分比）/（纯化前细胞总数×纯化前阳性细胞百分比）。6.人毛囊DPCs分离、培养。将含有毛囊中下部的皮下组织剪碎,在37℃缓慢搅拌的条件下加入胶原酶D 37℃孵育6—8h,悬液经400目筛网过滤,收集被筛网截留的毛乳头,PBS离心洗涤三次。放入25mL培养瓶中,加入DMEM/F12补充培养基于37℃、5%CO₂孵箱中培养,每3d换液,观察细胞增殖能力及形态学特征并描绘生长曲线。7.尝试毛囊重建。将纯化后获得的人HFSCs和人毛囊DPCs混合,细胞数比例为1:2,混合后细胞密度为1×10⁴/μL,200μL为一单位,注射入裸鼠皮下,PBS液为对照组,21d后进行组织切片。结果:1.通过毛囊解剖可观测到鼠与人毛囊的Bulge区均位于皮肤的皮脂腺与立毛肌这一层次。鼠的较为宽大,所含细胞较多;人的较为细小单薄,取材上可能较为困难。2.比较显微分离培养法和消化法发现,前者培养的鼠HFBCs,贴壁率高,细胞损失少,有利于细胞取材。3.用显微分离培养法获得的人HFBCs爬出速度快,细胞生长迅速,细胞消化后可见所获得的HFBCs并不均匀,为多种细胞混合。K19检测结果显示阳性。4.纯化后获得的HFSCs,荧光显微镜下观察可见细胞纯度提高,活性约为94%左右,生长迅速。5.HFSCs纯度及回收率结果显示,纯化前CD200阳性细胞的纯度为8.31%,纯化后CD200阳性细胞纯度为82.31%,阴性对照为1.63%。细胞纯化后,CD200阳性细胞回收率为65.39%。6.HFSCs细胞培养,第1、2天生长缓慢,第3天开始迅速增殖,第7天左右进入平台期,此时开始出现接触抑制,细胞增长缓慢,继续培养,10天左右细胞开始出现老化。7.裸鼠皮下注射人HFSCs和人毛囊DPCs混合物,21d后组织切片可见有新生毛囊样结构形成。结论:本实验联用了显微分离培养与免疫磁珠法,分离纯化人HFSCs。由于显微解剖法可首先从解剖学角度获得HFSCs富集部位,同时结合免疫磁珠法选择CD200阳性表达细胞,最终获得HFSCs。为其大量纯化及后续研究提供了新的备选技术路线。此方法较传统方法有操作简便、经济,能与细胞培养、流式细胞术、荧光显微镜等分子生物学技术兼容,单次操作分离细胞数量大且纯度较高的特点。所获得的HFSCs增殖迅速,与人毛囊DPCs混合可在一定条件下重新诱导生成毛囊样结构。更多还原

【Abstract】 Background:Nowadays, hair restoration surgery technique, although its curative effect affirmation, hold out for long time, it got a large number of bald-headed sufferer’s approve.But no matter which repair technique of adoption, all with the sufferer own to amplely provide hair area from the body for premise. For those sufferer whose bald rea area is too big or the even hair complete shattered, these repair of the technique often can’t use. Even these techniques are used, under the effect of the shortage of providing area and the transplant rate unsteady, the hair density after operation can’t attain satisfaction. Besides none surgical operation can creat new hair and can’t increase the amount of hair. Many bald-headed sufferer’s hair is losing every day, hair restoration surgery technique can’t make it stop, either, can’t estimate it develop circumstance. So, the shortage of the resources of hair area is obvious and become the present hair repair surgery more difficult problem for solve. And also to be a hurry request for the development of the hair follicle engineering technology.Hair follicle is the most complicated skin organization, crossing epidermis and dermis.It is structure is very different than other organization, and has a special immunity.Hair follicle is the result of the interaction between epidermis and dermis. In the embryo, the skin begins as a single layer of epidermal stem cells.Soon after, as mesenchymal cells populate the skin to form the underlying collagenous dermis, morphogenesis of the hair follicle begins. Hair follicle stem cells （HFSCs） can indeed influence hair growth. Therefore, there is most important to establish the HFSCs’ development model.And it will help to the study of the hair follicle morphogenesis and hair follicle switch from the vellus to the terminal state.On the other hand, we have to face the purification of the stem cell during the studying the stem cell. The previous belief that stem cells reside in the bulbar region of hair follicles.But the old method is hard to manipulate, cells are hard to adhere and grow slowly, can’t get the pure stem cells,it is also easy to be contaminated , this is a laboring task.Objective:1. To confirm the fixed position of the HFSCs.Determinate and improve the method of how to isolate and culture HFBCs.2. To confirm the HFSCs surface marks and attempts seeks the method of how to purifies HFSCs3. To establish the method of reconstructing hair follicles by mixes the HFSCswhich after purification and dermal papilla cells （DPCs）.Methods:1. The microdissection of mouse hair follicle and human hair follicle. Separates the mouse and person’s complete hair-follicle by microdissection.Put them under the inverted microscope to observe the difference of their shape and structure.2. Mouse HFBCs’ separation and cultivation.Micromanipulation:takes Wistar big mouse on 7th day age skin under the aseptic condition. After two rinses in D-Hanks’ contains penicillin and streptomycin. And the skin fragments were incubated in 0.25% dispase for 2h at 4℃. Those in anagen phase, were carefully selected under the dissecting microscope. After two rinses, then the bulge region was amputated from upper follicle by making two transversal cuts respectively at the site of the enlargement spots of bulge area with a fine needle.And immerse them in a supplemented mixture medium of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium （DMEM/F12） containing 10% fetal bovine serum as described. Enzyme digestion: put the bulge region which was got by micromanipulation in to 0.25% dispase mixed with 0.1% collagen enzyme for 20min at 37℃.Then blow the organization into cells.Collect the cells to the supplemented mixture medium of DMEM/F12 containing 10% fetal bovine serum to stop digesting.Then immersed in a DMEM/F12.The culture was incubated at 37℃and 5% CO₂ in air, and the medium changed twice a week.3. Human HFBCs’ separation and cultivation.Plastic surgery specimens of human scalp skin were obtained from the nape of the neck. After rinsing with 70% alcohol, the tissues were trimmed into small pieces, and the skin fragments were incubated in 0.25% dispase for 2h at 37℃. The hair follicles were drawed out form the side of dermis carefully. Those in anagen phase, were carefully selected under the dissecting microscope. After two rinses, then the bulge region was amputated from upper follicle by making two transversal cuts respectively at the site of the enlargement spots of bulge area with a fine needle. All surgical procedures were operated under a sterile environment. After additional two rinses, them were transferred into a new the follicles were transferred into a 25mL bottle, immersed in a supplemented mixture medium of DMEM/F12 containing 10% fetal bovine serum as described. The culture was incubated at 37℃and 5% CO₂ in air, and the medium changed twice a week.4. Human HFBCs’ examination and purification. Takes few HFBCs to put in the culture dish, After pasteing the wall in 48 hours. Then them were rinsed 3 times by PBS （each time 5min）. Precooling acetone fixed 10min. Immunohistochemical staining. Examined the K19’s expression. Float the HFBCs again.Adjust the density for 1×10⁸/mL, according to the density of 1.0μg/mL, immersed in FITC-mouse-anti-person CD200 30min.Separate the HFSCs by magnetic cell sorting.5. Human HFSCs’ examination. Float the purify cell again with 0.4% trypan bule.Then the viability of these purified HFSCs was detected under light microscope. The purification rate was analyzed by flow cytometry. The preand post-purification cells were compared by immunofluorescence staining.6. DPCs of human scalp hair follicles were isolated by digesting-filtrating method, and cultured in supplemented mixture medium of DMEM/F12 containing 10% fetal bovine serum as described.7. The mixture of HFSCs and DPCs were implanted onto the back skin of nude mouse by hypodermal injection.The histologic examination of the skin was performed after 21 days.Results:1. Both mouse and human’s bulge area were detected in the contiguous part of bulge area, that provides the insertion point for arrector pili muscle and marks the bottom of the permanent portion of hair follicles. In mouse pelage hair follicles, the bulge can be detected as discrete protuberances of the outer root sheath. Although the bulge can also be detected as a clearly defined structure in embryonic hair follicles.2. Compared with micromanipulation and enzyme digestion. In the former method, the adhering rate was high.It can’t waste too much cells.3. Cells proliferated rapidly in vitro by micromanipulation.Floated the cells again,the HFBCs were mixed with the other cells.4. After purification, the viability of cells was not significantly impaired. And the cells proliferated rapidly.5. Flow cytometry and immunofluorescence staining examination, the CD200⁺ cell rate was 8.31% before cell sorting purification while that was 82.31% after cell sorting purification.6. The earliest HFSCs was observed within 2 days after inoculation.Cells proliferated rapidly after 3 days.Then kept rapid growth until day 7.The cells began to decelerate after day 10.7. New formed hair follicles can be seen under microscopy after 21 days.Conclusion:Highly purified and viable human hair foll icle stem cells could be obtained by micromanipulation and magnetic cell sorting assay. This method can isolate HFSCs of human scalp hair follicless on a large scale rapidly and efficiently.The mixture of HFSCs and DPCs can form new hair follicles under particular conditions.更多还原