本文選題：毛囊隆突區(qū)細胞 + 毛囊干細胞　； 參考：《南方醫(yī)科大學》2008年碩士論文

【摘要】： 背景: 現(xiàn)代毛發(fā)修復外科技術,雖然其療效肯定、持久,曾得到廣大禿發(fā)患者的認可,但實踐證明無論是哪一種修復技術的采用,都是以患者自體擁有充足的供區(qū)毛發(fā)為前提。對于禿區(qū)面積大,甚至毛發(fā)完全缺失的患者,這些修復技術常無法開展;即便開展,由于供區(qū)不足、移植成活率不穩(wěn)定等因素,手術后的毛發(fā)密度也無法達到滿意效果。況且任何外科手術都不會產生新的毛發(fā),也不會增加毛發(fā)的數(shù)量。許多禿發(fā)患者毛發(fā)的丟失是進行性的,毛發(fā)修復也不能逆轉或停止其丟失,更無法預測其發(fā)展情況。所以,供區(qū)毛發(fā)資源明顯不足,成為現(xiàn)今毛發(fā)修復外科較難解決的問題,也對毛囊的組織工程技術快速發(fā)展提出了要求。 毛囊是上皮組織中最復雜的器官,橫跨表皮和真皮,不但其結構生理方面有很多特殊之處,而且有一套獨特的免疫方式。毛囊是上皮與間充質相互作用的結果,由分別來源于中外兩個胚層的上皮細胞和真皮細胞構成。毛囊的形態(tài)發(fā)生是細胞依附于細胞外基質在毛囊相關信號的誘導下定向移動與分化的結果,毛囊干細胞(HFSCs)對毛囊的形態(tài)發(fā)生和毛發(fā)的周期性生長起主導作用。因此建立HFSCs體外培養(yǎng)機制,對于詳盡地了解HFSCs體外培養(yǎng)生長特點,以及如何誘導毛囊的發(fā)生,具有重要的意義。 另一方面是干細胞研究必須要面對的干細胞純化問題,目前普遍認為HFSCs定位于毛囊上段的隆突(Bulge)區(qū),與基質具有高黏附性是HFSCs分離培養(yǎng)困難的重要因素之一。但目前傳統(tǒng)的分離方法,操作繁雜、勞動量大,同時獲得的毛囊隆突區(qū)細胞(HFBCs)中HFSCs純度不高,易污染,因而不便于實驗開展。 目的: 1.證實HFBCs在毛干上的定位,確定分離培養(yǎng)HFBCs的方法,并尋找技術改良。 2.證實HFSCs的表面標記并嘗試尋找純化HFSCs的方法。 3.進一步利用純化后的HFSCs和毛乳頭細胞(DPCs)混合進行體內誘導試驗,嘗試誘導出新生毛囊及毛發(fā)纖維。 方法: 1.鼠和人毛囊的顯微解剖。采用顯微解剖法分離出鼠及人的完整毛囊。將其置于倒置顯微鏡下觀察其形態(tài)結構和區(qū)別。 2.鼠HFBCs的分離、培養(yǎng)。顯微分離培養(yǎng)法:無菌條件下取7日齡Wistar大鼠觸須部皮膚,在含雙抗(青鏈霉素)的D-Hanks'液中清洗2遍,在體視顯微鏡下用27號針頭將毛囊從組織中分離出來,置于0.25%Dispase酶中,4℃消化2h后用D-Hanks'液清洗,在體視顯微鏡下輕輕擠壓毛球部即可將毛囊Bulge區(qū)及毛干完整地分離出來,切去皮脂腺及毛囊中下段,保留Bulge區(qū)毛囊,將其置于含10%(體積比)胎牛血清的DMEM/F12補充培養(yǎng)基中進行培養(yǎng)。消化法:將組織法獲得的毛囊Bulge區(qū)置于含0.25%Dispase酶和0.1%膠原酶的混合消化液中,37℃消化20min,吹打制懸,吸取懸液至離心管中并加入含血清培養(yǎng)基終止消化,剩余組織塊繼續(xù)加入消化液消化,并分次采集細胞懸液,直至組織塊消化完全。收集離心后得到的細胞,并加入含血清培養(yǎng)基再次離心,最后加入含10%(體積比)胎牛血清的DMEM/F12補充培養(yǎng)基制成單細胞懸液,接種到培養(yǎng)瓶中。觀察并比較兩種方法獲得的鼠HFBCs在DMEM/F12補充培養(yǎng)基中體外培養(yǎng)的形態(tài)學和生物學特點。 3.人HFBCs的分離、培養(yǎng)。取頭皮標本,參照顯微分離培養(yǎng)法并加以改進,備皮后于70%乙醇中清洗,盡量除去皮下脂肪。將皮片切成長條,置入0.25%Dispase酶37℃消化2h。從真皮-皮下組織交界處橫斷頭皮,用鑷子從皮下組織端拉出毛囊,收集形態(tài)完好且處于生長期的毛囊,體視顯微鏡下分別在球部上端、皮脂腺下端橫切毛囊,取中間部分,含雙抗PBS漂洗3次,放入25mL培養(yǎng)瓶中,加入DMEM/F12補充培養(yǎng)基于37℃、5%CO_2孵箱中培養(yǎng),每3d換液,觀察細胞增殖能力及形態(tài)學特征。 4.人HFSCs的鑒定與純化。取少量HFBCs置于培養(yǎng)皿中,待其48小時重新貼壁后,經PBS漂洗3次(每次5min),預冷丙酮固定10min,然后常規(guī)免疫細胞化學方法染色,檢測K19的表達情況,以PBS代替一抗作陰性對照。將HFBCs重懸,調整為1×10~8/mL,按1.0μg/mL濃度,加入FITC標記的鼠抗人CD200單抗,室溫孵育30min。離心棄上清中未結合的抗體。加入1mL磁珠分選緩沖液重懸細胞,離心棄上清,PBS緩沖液清洗2次。取重懸后HFBCs與50μg免疫磁珠混合,室溫反應30min,磁珠分離器分離8min,去除未與磁珠結合的細胞。將結合了靶細胞的免疫磁珠用1mL磁珠分選緩沖液洗5次。37℃、5%CO_2孵箱培養(yǎng)48h,使細胞與磁珠分離,得到較純的HFSCs。 5.人HFSCs的檢測。細胞活性檢測:將純化后細胞懸液與0.4%苔盼藍溶液按1:1體積比混合后,滴入細胞計數(shù)板置于顯微鏡下觀察。細胞免疫熒光檢測:分別于純化前后取5μL細胞懸液于熒光顯微鏡下觀察其表達CD200情況。HFSCs純度及回收率分析:取CD200孵育后行磁珠分選前后的HFBCs進行實驗,以對照試劑室溫孵育30min的HFBCs為對照。各取5×10~5個細胞上機。以對照管作為空白標定,記錄標本的CD200陽性細胞百分率。流式數(shù)據(jù)采用Cellquest pro分析。純度=陽性細胞數(shù)/細胞總數(shù);回收率=(純化后細胞總數(shù)×純化后陽性細胞百分比)/(純化前細胞總數(shù)×純化前陽性細胞百分比)。 6.人毛囊DPCs分離、培養(yǎng)。將含有毛囊中下部的皮下組織剪碎,在37℃緩慢攪拌的條件下加入膠原酶D 37℃孵育6—8h,懸液經400目篩網過濾,收集被篩網截留的毛乳頭,PBS離心洗滌三次。放入25mL培養(yǎng)瓶中,加入DMEM/F12補充培養(yǎng)基于37℃、5%CO_2孵箱中培養(yǎng),每3d換液,觀察細胞增殖能力及形態(tài)學特征并描繪生長曲線。 7.嘗試毛囊重建。將純化后獲得的人HFSCs和人毛囊DPCs混合,細胞數(shù)比例為1:2,混合后細胞密度為1×10~4/μL,200μL為一單位,注射入裸鼠皮下,PBS液為對照組,21d后進行組織切片。 結果: 1.通過毛囊解剖可觀測到鼠與人毛囊的Bulge區(qū)均位于皮膚的皮脂腺與立毛肌這一層次。鼠的較為寬大,所含細胞較多;人的較為細小單薄,取材上可能較為困難。 2.比較顯微分離培養(yǎng)法和消化法發(fā)現(xiàn),前者培養(yǎng)的鼠HFBCs,貼壁率高,細胞損失少,有利于細胞取材。 3.用顯微分離培養(yǎng)法獲得的人HFBCs爬出速度快,細胞生長迅速,細胞消化后可見所獲得的HFBCs并不均勻,為多種細胞混合。K19檢測結果顯示陽性。 4.純化后獲得的HFSCs,熒光顯微鏡下觀察可見細胞純度提高,活性約為94%左右,生長迅速。 5.HFSCs純度及回收率結果顯示,純化前CD200陽性細胞的純度為8.31%,純化后CD200陽性細胞純度為82.31%,陰性對照為1.63%。細胞純化后,CD200陽性細胞回收率為65.39%。 6.HFSCs細胞培養(yǎng),第1、2天生長緩慢,第3天開始迅速增殖,第7天左右進入平臺期,此時開始出現(xiàn)接觸抑制,細胞增長緩慢,繼續(xù)培養(yǎng),10天左右細胞開始出現(xiàn)老化。 7.裸鼠皮下注射人HFSCs和人毛囊DPCs混合物,21d后組織切片可見有新生毛囊樣結構形成。 結論: 本實驗聯(lián)用了顯微分離培養(yǎng)與免疫磁珠法,分離純化人HFSCs。由于顯微解剖法可首先從解剖學角度獲得HFSCs富集部位,同時結合免疫磁珠法選擇CD200陽性表達細胞,最終獲得HFSCs。為其大量純化及后續(xù)研究提供了新的備選技術路線。此方法較傳統(tǒng)方法有操作簡便、經濟,能與細胞培養(yǎng)、流式細胞術、熒光顯微鏡等分子生物學技術兼容,單次操作分離細胞數(shù)量大且純度較高的特點。所獲得的HFSCs增殖迅速,與人毛囊DPCs混合可在一定條件下重新誘導生成毛囊樣結構。
[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.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R329

【參考文獻】

相關期刊論文 前7條

1 仇文穎,許增祿;毛囊干細胞的研究進展[J];基礎醫(yī)學與臨床;2002年04期

2 李宇,林常敏,蔡湘娜,李國強;微囊法體外重建人頭皮毛乳頭誘導毛囊再生功能的評價[J];中國臨床康復;2005年06期

3 孫曉慶,付小兵,盛志勇;表皮干細胞[J];中華創(chuàng)傷雜志;2000年10期

4 劉榮卿,鐘白玉,唐書謙,葉慶佾,呂中法,伍津津;裸鼠重建毛囊的組織學研究[J];中華皮膚科雜志;2000年04期

5 呂中法;蔡綏R，

本文編號：2103368