毛囊干细胞标记与纯化的相关研究

发布时间：2018-07-06 16:06

本文选题：毛囊隆突区细胞 + 毛囊干细胞　；参考：《南方医科大学》2008年硕士论文

【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%CO_2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1×10~8/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%CO_2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5×10~5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%CO_2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1×10~4/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。
[Abstract]:Background:
Modern hair repair surgery technology, although its curative effect, lasting, has been recognized by the vast number of bald patients, but the practice has proved that no matter which kind of repair technology is used, the patient has sufficient donor hair as the premise. For the patients with large area of bald area and even complete hair loss, these repair techniques are often unable to carry out. Even if it is carried out, the density of hair after the operation is not satisfactory because of the shortage of the donor area and the instability of the survival rate. Moreover, any surgical operation will not produce new hair or increase the amount of hair. The loss of hair in many bald patients is progressive, and the hair repair can not reverse or stop the loss of hair. It is more difficult to predict its development. Therefore, the shortage of hair resources in the donor area has become a difficult problem to be solved in the current hair repair surgery, and it also demands the rapid development of hair follicle tissue engineering technology.
The hair follicle is the most complex organ in the epithelial tissue, spanning the epidermis and dermis, not only its structural and physiological aspects, but also a unique set of immune methods. The hair follicle is the result of the interaction between the epithelium and the mesenchyme, which is composed of the epithelial cells and dermis derived from two germ layers in China and foreign countries. The morphology of the hair follicles is the form of the hair follicle. Cells adhere to the results of directional movement and differentiation of the extracellular matrix under the induction of hair follicle related signals. Hair follicle stem cells (HFSCs) play a leading role in the morphogenesis of hair follicles and the periodic growth of hair. Therefore, the mechanism of HFSCs culture in vitro is established to understand the growth characteristics of HFSCs in vitro, and how to induce hair follicle. It is of great significance.
On the other hand, stem cell purification must face the problem of stem cell purification. At present, it is generally believed that HFSCs is located in the Bulge region of the upper part of the hair follicle, and the high adhesion of the matrix is one of the important factors for the difficulty of the separation and culture of HFSCs. However, the traditional method of separation is complicated, the amount of labor is large and the bursa area of the hair follicle is fine. The purity of HFSCs in cell (HFBCs) is not high and easy to pollute, so it is not easy to carry out experiments.
Objective:
1. confirm the location of HFBCs on the stem, identify the method of isolation and culture of HFBCs, and seek for technical improvement.
2. confirm the surface marker of HFSCs and try to find a method to purify HFSCs.
3. further use of purified HFSCs and dermal papilla cells (DPCs) to induce in vivo induction of new hair follicles and hair fibers.
Method:
1. the microanatomy of mouse and human hair follicles. The intact hair follicles of mice and humans were separated by microdissection. The morphology and structure of the hair follicles were observed under inverted microscope.
2. rat HFBCs isolation and culture. Microisolation culture method: under aseptic condition, the skin of the tentacle part of 7 day old Wistar rats was removed for 2 times in the D-Hanks'solution containing double resistance (penicillin), and the hair follicle was separated from the tissue with the 27 needle under the stereoscopic microscope, and was placed in the 0.25% Dispase enzyme. After digestion for 2h at 4 C, the hair follicle was cleaned with D-Hanks' solution and displayed in the stereopsis. The hair follicle Bulge area and hairy stem can be completely separated from the hair bulb under the micromirror, and the sebaceous glands and the middle and lower segments of the hair follicle are cut off, and the hair follicles in the Bulge area are retained and placed in the DMEM/F12 supplemental medium containing 10% (volume ratio) fetal bovine serum. The digestion method is placed in the 0.25%Dispase enzyme and 0.1% of the hair follicles obtained by tissue method. In the mixed digestion solution of collagenase, 20min was digested at 37 degrees C, the suspension was blown, the suspension was absorbed into the centrifuge tube and the serum containing medium was added to terminate the digestion. The remaining tissue blocks continued to be digested with the digestive juice, and the cell suspension was collected, until the tissue block was digested completely. The cells obtained after the centrifuge were collected and centrifuged again with a serum medium medium. In the end, a single cell suspension was made into a single cell suspension containing 10% (volume ratio) fetal bovine serum DMEM/F12 and inoculated into the culture bottle. The morphological and biological characteristics of the rat HFBCs in the DMEM/F12 supplemental medium were observed and compared with the two methods.
3. people HFBCs separation, culture. Take the scalp specimen, refer to the microseparation culture method and improve it. After preparing the skin, the skin is cleaned in 70% ethanol, and the subcutaneous fat is removed. The skin slices are cut into strips, and the 0.25%Dispase enzyme is digested at 37 C to digest the scalp from the dermis subcutaneous tissue junction and pull out the hair follicle from the subcutaneous tissue end with tweezers and collect the form. A good and growing hair follicle, under the stereoscopic microscope, at the upper end of the sphere, the lower end of the sebaceous gland, the hair follicle, the middle part, the double anti PBS rinse 3 times, into the 25mL culture bottle, and the DMEM/F12 supplement culture based on 37 degrees C, the incubator in 5%CO_2 and the change of each 3D, to observe the cell proliferation and morphological characteristics.
4. people HFSCs was identified and purified. A small amount of HFBCs was placed in a Petri dish. After 48 hours of reattachment, 3 times (5min) were rinsed through PBS and 10min was fixed by precooled acetone. The expression of K19 was detected by routine immunocytochemical staining, and PBS was used instead of a negative control. HFBCs was suspended and adjusted to 1 * 10~8/mL, 1 u g/mL concentration. At room temperature, the anti human CD200 mAb was incubated with FITC, and the unconjugated antibody in the supernatant was centrifuged at room temperature. Adding 1mL magnetic beads to separate the suspension cells, centrifuging the supernatant and cleaning the PBS buffer for 2 times. The HFBCs and the 50 g immunomagnetic beads were mixed, the room temperature reaction 30min, the magnetic bead separator separated 8min, and the separation of the non magnetic beads from the magnetic beads. The immunomagnetic beads of target cells were washed with 1mL magnetic beads for 5 times.37 degrees, and incubated in 5%CO_2 incubator for 48h.
The detection of 5. people HFSCs. Cell activity detection: after the purified cell suspension and 0.4% moss blue solution were mixed with the 1:1 volume ratio, the cell count plate was observed under the microscope. Cell immunofluorescence detection: the purity and recovery of the.HFSCs expression of CD200 were observed under the fluorescence microscope before and after the purification, respectively. The HFBCs of the magnetic bead sorting before and after CD200 incubation was tested with the control reagent at room temperature incubated with 30min HFBCs as the control. Each 5 x 10~5 cells were taken on the machine. The percentage of CD200 positive cells was recorded as a blank. The flow data was analyzed by Cellquest pro. The purity = positive cell number / total number of cells; recovery = (pure) = (pure) The total number of positive cells after purification (percentage of purified cells) / (the total number of cells before purification * the percentage of positive cells before purification).
The 6. hair follicle DPCs was separated and cultured. The subcutaneous tissue containing the middle and lower parts of the hair follicle was shredded, and 6 8h was incubated with collagenase D 37 C under the condition of slow stirring at 37 degrees C. The suspension was filtered by 400 mesh screen, and the hair nipple was collected by the sieve, and PBS centrifuged for three times. In the 25mL culture bottle, the DMEM/F12 supplemented culture was based on 37, 5%CO_2 incubator. In medium culture, the proliferation ability and morphological characteristics of each 3D were observed and the growth curve was characterized.
7. to try the hair follicle reconstruction. The purified human HFSCs and human hair follicle DPCs were mixed, the number of cells was 1:2, the cell density was 1 x 10~4/ mu L, 200 mu L was one unit, the subcutaneous injection into nude mice, the PBS solution as the control group, and the tissue section after 21d.
Result:
1. the Bulge area of the rat and human hair follicle can be observed by the hair follicle. The sebaceous glands and the muscle of the skin are located at the level of the sebaceous gland and the muscle of the skin. The mice are more broad and contain more cells.
2. compared with microscopic isolation and digestion, it was found that mouse HFBCs cultured in vitro had higher adherence rate and less cell loss, which was beneficial to cell extraction.
3. the human HFBCs obtained by microdissection culture can climb out quickly, the cell growth is rapid, and the HFBCs obtained after the digestion of the cells is not uniform, which is positive for the results of a variety of cells mixed with.K19.
4. after purification, HFSCs was observed under fluorescence microscope. The purity of the cells was increased, the activity was about 94%, and the growth was rapid.
The purity and recovery of 5.HFSCs showed that the purity of CD200 positive cells before purification was 8.31%, the purity of the purified CD200 positive cells after purification was 82.31%, and the negative control was purified by 1.63%. cells, and the recovery rate of CD200 positive cells was 65.39%.
6.HFSCs cell culture, the 1,2 was born slowly, began to proliferate quickly on the third day and entered the platform period of about seventh days. At this time, the contact inhibition began to appear, the cell growth was slow, and the cell began to grow, and the cells began to appear aging around 10 days.
7. the mixture of human HFSCs and human hair follicle DPCs was injected subcutaneously into nude mice. After 21d, new hair follicle like structure was seen in the tissue sections.
Conclusion:
In this experiment, the microseparation culture and immunomagnetic beads were used in this experiment. The separation and purification of human HFSCs. could first obtain the HFSCs enrichment site from the anatomical point of view. At the same time, the immunomagnetic bead method was used to select the CD200 positive cells. Finally, a new technical route was provided for the purification and subsequent research of HFSCs.. Compared with traditional methods, it is easy to operate, economy, compatible with cell culture, flow cytometry, fluorescence microscopy and other molecular biological techniques. The single operation is large and high in purity. The obtained HFSCs proliferation is rapid, and the mixture of human hair follicle DPCs can be re induced to produce hair follicle like structure under certain conditions.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

【参考文献】