本文選題：毛囊干細(xì)胞 + Notch信號(hào)�。� 參考：《復(fù)旦大學(xué)》2007年博士論文

【摘要】： 第一部分小鼠鼠須及人頭皮毛囊干細(xì)胞的定位及特點(diǎn) 目的明確小鼠鼠須墊毛囊及成年男性額部毛囊干細(xì)胞的定位及分布特點(diǎn)，篩選鼠須墊切片快速高效的方法，比較正常成年男性與脂溢性脫發(fā)成年男性額部毛囊干細(xì)胞數(shù)量差別。 方法取8周齡BALB／C小鼠雙側(cè)鼠須墊，自制標(biāo)本固定器脫水，包埋切片，作常規(guī)HE染色及角蛋白15(CK15)免疫組化；取正常成年男性及脂溢性脫發(fā)成年男性額部頭皮，脫水包埋切片，并作常規(guī)HE染色及角蛋白15免疫組化，顯微鏡下分析結(jié)果并比較。 結(jié)果在顯微鏡下：①HE染色及CK15免疫組化能明確顯示小鼠(BALB／C)鼠須墊毛囊毛囊外根鞘(ORS)、內(nèi)跟鞘(IRS)、毛乳頭(FP)及毛干(HS)及立毛肌等結(jié)構(gòu)。在毛囊立毛肌止點(diǎn)外根鞘隆起的部位小范圍角蛋白CK15表達(dá)明顯。②HE染色及CK15免疫組化能明確顯示成年男性額部頭皮毛囊外根鞘、內(nèi)跟鞘、毛乳頭、皮脂腺、毛干及立毛肌等結(jié)構(gòu)。但與小鼠不同，成年男性額部頭皮毛囊CK15表達(dá)自皮脂腺開口水平以下的外根鞘狹長區(qū)域延展至中部，逐漸變淡。額部毛囊干細(xì)胞數(shù)量正常成年男性高于脂溢性脫發(fā)成年男性(P＜0.05)。 結(jié)論小鼠鼠須毛囊干細(xì)胞位于毛囊立毛肌止點(diǎn)附近的隆突內(nèi)，人頭皮毛囊干細(xì)胞位于毛囊皮脂腺開口水平以下狹長區(qū)域的外根鞘內(nèi)，并且額部毛囊干細(xì)胞數(shù)量正常成年男性高于脂溢性脫發(fā)成年男性。 第二部分小鼠鼠須毛囊干細(xì)胞向短暫增殖細(xì)胞(TA細(xì)胞)分化的研究 目的研究小鼠鼠須毛囊干細(xì)胞在外源性表皮生長因子(EGF)及創(chuàng)傷的誘導(dǎo)下，向TA細(xì)胞分化的情況。 方法將8周齡BALB／C小鼠隨機(jī)分4組，雙側(cè)鼠須墊去表皮，每天經(jīng)口內(nèi)頰粘膜注射EGF，分別為OIU，1000IU，5000IU，10000IU，觀察創(chuàng)面恢復(fù)情況，確定最佳干預(yù)劑量。 將8周齡BALB／C小鼠隨機(jī)分三組，分別記為EGF組、創(chuàng)傷組和空白組。EGF干預(yù)采用每天經(jīng)口內(nèi)頰粘膜注射法(劑量5000IU)，創(chuàng)傷采用鼠須墊去表皮法。①分別于D0、D1、D3、D5、D7、D9(D-天)收集鼠須墊標(biāo)本，脫水固定，做CK15及CK14免疫組化，CK15及CK14陽性細(xì)胞計(jì)數(shù)，方差分析不同組別不同時(shí)間有無差別。②取三組D0、D1、D3、D5、D7、D9小鼠鼠須墊，提取其毛囊真皮鞘細(xì)胞，F(xiàn)ACS分析其CK15及CK14陽性細(xì)胞比率變化。 結(jié)果三種劑量EGF均加速傷口愈合，OIUEGF組創(chuàng)面愈合時(shí)間為9天，1000IUEGF組為8天，5000IUEGF及10000IU組沒有差別，均為7天。確定每天一次給予5000IU EGF為后續(xù)實(shí)驗(yàn)干預(yù)劑量。 免疫組化結(jié)果：①CK15陽性細(xì)胞聚集于隆突部，與第一部分實(shí)驗(yàn)結(jié)果一致：a同組別不同時(shí)點(diǎn)比較，EGF組在不同時(shí)點(diǎn)CK15陽性細(xì)胞數(shù)目差別顯著。第1天比第0天明顯減少，第3天比第1天明顯減少，第5天比第3天明顯減少，第7天比第5天明顯減少，第9天比第7天明顯減少(均為P＜0.05)；創(chuàng)傷組在不同時(shí)間點(diǎn)CK15陽性細(xì)胞數(shù)目也呈現(xiàn)類似的變化，CK15陽性細(xì)胞漸次減少(均為P＜0.05)；空白組在不同時(shí)間CK15陽性細(xì)胞數(shù)目無明顯變化(P＞0.05)。b同時(shí)點(diǎn)不同組別比較，在第0天時(shí)各組CK15陽性細(xì)胞數(shù)目無顯著差別(P＞0.05)，在第1天，EGF組及創(chuàng)傷組與空白組都有明顯差別(P＜0.05)，但兩者之間無顯著差別(P＞0.05)。第3，5，7，9天不同組別CK15陽性細(xì)胞數(shù)目差別顯著，均為第空白組最多，其次為創(chuàng)傷組，EGF組組最少(均為P＜0.05)。②CK14陽性細(xì)胞分布范圍則明顯廣于CK15陽性細(xì)胞，毛囊外根鞘及皮下組織均可見廣泛表達(dá)：a同組別不同時(shí)點(diǎn)比較，EGF組在不同時(shí)點(diǎn)CK14陽性細(xì)胞數(shù)目差別顯著。第1天比第0天明顯增多，第1到7天緩慢增多，第9天比第7天又明顯增多(均為P＜0.05)；創(chuàng)傷組在不同時(shí)間CK14陽性細(xì)胞細(xì)胞數(shù)目也不同，增多速度比較平緩；空白組在不同時(shí)間CK14陽性細(xì)胞數(shù)目無明顯變化(P＞0.05)。b同時(shí)點(diǎn)不同組別比較，在第0天時(shí)各組CK14陽性細(xì)胞數(shù)目無顯著差別(P＞0.05)，第1，3，5，7，9天不同組別CK14陽性細(xì)胞數(shù)目差別顯著，均為空白組最少，其次為創(chuàng)傷組，EGF組最多(均為P＜0.05)。 流式細(xì)胞分析結(jié)果：分析顯示EGF組、創(chuàng)傷組CK15陽性細(xì)胞比率從第1天開始就明顯下降，且下降速率前者大于后者，空白組沒有明顯變化。EGF組、創(chuàng)傷組CK14陽性細(xì)胞比率逐漸增加，且前者大于后者，空白組沒有明顯變化。 結(jié)論①外源性EGF能加速小鼠創(chuàng)面的愈合，每天給予5000IU能顯著加速創(chuàng)面的愈合。 ②EGF與創(chuàng)傷均能啟動(dòng)小鼠鼠須毛囊干細(xì)胞向TA細(xì)胞分化。EGF能加速創(chuàng)面的愈合，這種作用在干預(yù)的前期效果明顯，隨著時(shí)間的推移，作用減弱。 第三部分EGF干預(yù)下小鼠鼠須毛囊外根鞘Notch信號(hào)變化的研究 目的研究在外源性EGF及創(chuàng)傷的刺激下，小鼠毛囊外根鞘細(xì)胞中Notch信號(hào)的相關(guān)組分變化情況及信號(hào)的活化情況。 方法將8周齡BALB／C小鼠均分三組，分別為EGF組、創(chuàng)傷組和空白組。EGF干預(yù)采用每天間斷經(jīng)口內(nèi)頰粘膜注射法，創(chuàng)傷采用鼠須墊去表皮法。分別于D0、D1、D3、D5(D-天)收集鼠須墊標(biāo)本，分離毛囊外根鞘，制成單細(xì)胞懸液，抽取mRNA，逆轉(zhuǎn)錄成cDNA，RT-PCR分別做Notchl、Delta、EGFR、及Notch信號(hào)的活化成分ICN基因測(cè)試，并比較各組有無差別。 結(jié)果①各組細(xì)胞均能檢測(cè)到Notchl、Delta、EGFR基因，且條帶半定量分析基本相同，無明顯區(qū)別(P＞0.05)。②EGF干預(yù)組在第1天能檢測(cè)到ICN條帶，灰度較淡。第1，3，5天均未測(cè)得。另外兩組標(biāo)本均未能測(cè)得ICN條帶。 結(jié)論①在外源性EGF刺激下，小鼠鼠須毛囊外根鞘細(xì)胞Notch信號(hào)被激活，且早期明顯。提示EGF通過Notch信號(hào)途徑促進(jìn)創(chuàng)面的愈合。 ②創(chuàng)傷刺激未能激活Notch信號(hào)。 第四部分人自體毛囊移植治療慢性大面積創(chuàng)面的研究 目的探索用自體毛囊移植治療慢性大面積創(chuàng)面的可能性。 方法選擇慢性難治性大面積創(chuàng)面病例，患者知情同意，創(chuàng)面清創(chuàng)，控制感染至新鮮肉芽組織無膿性滲液。枕后備皮，英國刀取刃厚皮，修剪成0.5cm×0.5cm郵票狀；切取取皮區(qū)毛囊，顯微鏡下單根分離約400根，形成去表皮毛囊。創(chuàng)面縱行分為兩區(qū)：毛囊種植區(qū)及刃厚植皮區(qū)。凡士林油紗布覆蓋，抗生素生理鹽水紗布包扎，患肢制動(dòng)。定期換藥，觀察兩區(qū)創(chuàng)面愈合情況；取活檢，HE常規(guī)染色，免疫組化檢測(cè)CK廣譜、vimentin(彈性蛋白)表達(dá)，觀察新生皮膚情況。 結(jié)果①大體觀察術(shù)后2周：毛囊種植區(qū)可以看到蒼白色新生上皮由移植毛囊向外長出，，以移植毛囊為中心成鵝卵石皮島樣分布；刃厚植皮區(qū)植皮成活，但生長較慢。術(shù)后8周：毛囊種植區(qū)新生皮膚連接成片，色澤基本一致，并有部分新生汗毛樣纖細(xì)毛發(fā)長出；刃厚植皮區(qū)瘢痕化明顯，皮膚色澤不均。術(shù)后10周：毛囊種植區(qū)新生皮膚逐漸成熟，成暗紅色，能耐受一定程度摩擦；刃厚植皮區(qū)瘢痕化明顯，凹凸不平。術(shù)后4月：毛囊種植區(qū)新生皮膚成熟，顏色質(zhì)地均勻，并有纖細(xì)汗毛樣毛發(fā)生長；刃厚植皮區(qū)則瘢痕明顯，質(zhì)地較差。②免疫組化毛囊種植區(qū)2周始新生表皮由毛囊延伸長出，CK廣譜表達(dá)較高，并出現(xiàn)表皮與真皮間的釘突結(jié)構(gòu)(rete ridges)，刃厚植皮區(qū)也看到類似結(jié)構(gòu)。8周后，毛囊種植區(qū)新生皮膚見到大量釘突樣結(jié)構(gòu)，表皮與其下真皮樣組織結(jié)合緊密，表達(dá)大量vimentin，未發(fā)現(xiàn)皮脂腺及汗腺結(jié)構(gòu)，刃厚植皮區(qū)也未發(fā)現(xiàn)腺體結(jié)構(gòu)。 結(jié)論①自體毛囊移植后表皮細(xì)胞以毛囊為中心向外長出，修復(fù)創(chuàng)面。愈合創(chuàng)面有一定耐磨擦能力。 ②對(duì)于慢性大面積難治性創(chuàng)面的治療，自體毛囊移植可以做為選擇的方法之一。
[Abstract]:The location and characteristics of the first part of mice and human scalp hair follicle stem cells
Objective to identify the location and distribution of mouse hair pad and adult male hair follicle stem cells, and screen a fast and efficient way to slice rat pad. Compare the number of hair follicle stem cells between adult male and seborrheic alopecia adult male.
Methods C / BALB mice 8 weeks old rats to bilateral pad, self-made specimen fixation and dehydration, embedded in paraffin, routine HE staining and cytokeratin 15 (CK15) immunohistochemistry; normal adult male and adult male seborrheic alopecia frontal scalp dehydration, embedded in paraffin, and routine HE staining and cytokeratin 15 immunohistochemical staining, microscope analysis results were compared.
Results: under the microscope of HE staining and CK15 immunohistochemistry can clearly display the mouse (BALB / C) mousewhisker pad hair follicle outer root sheath (ORS), with a sheath (IRS), the dermal papilla and hair stem (FP) (HS) and common structure. In the hair follicle outer root common stopping point a small range of sheath bulge the keratin CK15 expression significantly. HE staining and CK15 immunohistochemistry can clearly show the adult male frontal scalp hair follicle outer root sheath, with a sheath, dermal papilla, sebaceous gland, hair dry and arrector structure. But with different adult male mice, frontal scalp hair follicle CK15 expression since the opening of the outer root sheath of sebaceous glands below the level of the narrow area extending to the middle, gradually fades. The number of cells is higher than that of normal adult male seborrheic alopecia adult male forehead hair follicle stem (P < 0.05).
Conclusion mouse hair follicle stem cells in hair follicles to stop near the carina in common, cells in the hair follicle sebaceous gland openings below the level of the narrow region within the outer root sheath of human hair follicle stem, and frontal hair follicle stem cells in normal adult males is higher than that of adult male seborrheic alopecia.
Study on the differentiation of hair follicle stem cells from mouse mouse hair follicle to transient proliferating cell (TA cell) in second parts
Objective to study the differentiation of hair follicle stem cells from mouse mouse hair follicle to TA cells induced by exogenous epidermal growth factor (EGF) and trauma.
Methods 8 week old BALB / C mice were randomly divided into 4 groups: bilateral mouse pad and epidermis, EGF was injected into buccal mucosa every day, OIU, 1000IU, 5000IU and 10000IU respectively, and wound healing was observed, and the best intervention dose was determined.
C BALB / 8 week old mice were randomly divided into three groups, respectively EGF group, trauma group and the control group of.EGF treated with daily buccal mucosa injection method (dose 5000IU), using the mouse pad to be wound. The epidermal respectively at D0, D1, D3, D5, D7, D9 (D- day) were collected to pad specimens, dehydration fixed, CK15 and CK14 immunohistochemistry, CK15 and CK14 positive cell count, variance analysis of different groups in different time have no difference. The three groups were D0, D1, D3, D5, D7, D9 to extract the mouse pad, dermal sheath cells. FACS analysis of the CK15 and CK14 positive cell ratio changes.
Results the three doses of EGF accelerated wound healing. The wound healing time in group OIUEGF was 9 days, and the 1000IUEGF group was 8 days. There was no difference between 5000IUEGF group and 10000IU group, all of them were 7 days.
The results of immunohistochemistry: CK15 positive cells in the bulge, and the first part of the experimental results are consistent with the a group, EGF group significantly at different time point. The difference between the number of CK15 positive cells in first days were less than zeroth days, third days were less than first days, fifth days were less than third day, seventh days were less than fifth days, Ninth days were less than seventh days (P < 0.05); trauma group also showed a similar change in the number of different time points of CK15 positive cells, CK15 positive cells gradually decreased (P < 0.05); the control group no significant change in the number of different time of CK15 positive cells (P > 0.05).B at different groups, on the zeroth day when the number of CK15 positive cells were no significant difference (P > 0.05), in first days, EGF group and trauma group and blank group had significant difference (P < 0.05), but there was no significant difference between them (P > 0.05). Day 3,5,7,9 of different groups of the number of CK15 positive cells were significantly different, the blank group was the largest, followed by trauma group, EGF group at least (P < 0.05). The CK14 positive cells were significantly broader than the distribution range of CK15 positive cells, outer root sheath and subcutaneous tissue showed extensive expression: a the same group at different time points, EGF group significantly at different time point. The difference between the number of CK14 positive cells increased significantly compared to first days for zeroth days, first to 7 days of slow increase, Ninth days more than seventh days and increased significantly (P < 0.05); the number of different time in the trauma group CK14 positive cells are also different increase, speed is relatively flat; the blank group had no obvious change in the number of CK14 positive cells in different time (P > 0.05).B at different groups, on the zeroth day when the number of CK14 positive cells were no significant difference (P > 0.05), day 1,3,5,7,9 of different groups of the number of CK14 positive cells The difference was significant, all were the least in the blank group, and the second was the trauma group, and the EGF group was the most (P < 0.05).
Flow cytometry analysis results: the results showed that EGF group, trauma group the rate of CK15 positive cells from first day decreased significantly, and the decrease rate of the former than the latter, the blank group did not change significantly in.EGF group, trauma group the rate of CK14 positive cells increased gradually, and the former is larger than the latter, the blank group did not change significantly.
Conclusion (1) exogenous EGF can accelerate the healing of wound in mice, and 5000IU can significantly accelerate the healing of the wound.
EGF and trauma can activate mouse mouse hair follicle stem cells to differentiate into TA cells..EGF can accelerate wound healing. This effect is obvious in the early stage of intervention, and it decreases with time.
Study on the changes of Notch signal in the outer root sheath of mouse hair follicle under the intervention of third part EGF
Objective to study the changes in the related components of the Notch signal in the outer root sheath cells of the mouse hair follicle and the activation of the signal under the stimulation of exogenous EGF and trauma.
Methods C / BALB mice aged 8 weeks were divided into three groups, respectively EGF group, trauma group and the control group of.EGF intervention in the daily intermittent buccal mucosa injection method, using the mouse pad to be wound epidermis method. Respectively in D0, D1, D3, D5 (D- days) were collected to pad specimens. The separation of the outer root sheath, made into single cell suspension, mRNA extraction, reverse transcription into cDNA, Delta, RT-PCR respectively Notchl, EGFR, ICN and Notch gene activation component test signal, and compare the difference.
Results the gene cells of each group were detected Notchl, Delta, EGFR, and a semi quantitative analysis with basically the same, no significant difference (P > 0.05). The EGF group can be detected ICN bands in first days, the gray is light. The first 1,3,5 days were not measured. The other two groups not measured ICN bands.
Conclusion: (1) under exogenous EGF stimulation, Notch signaling in mouse hair follicle outer root sheath cells was activated, and early signiant. It suggests that EGF promotes the healing of wounds through Notch signaling pathway.
2. The Notch signal was not activated by the traumatic stimulation.
The study of fourth parts of autologous hair follicle transplantation for the treatment of chronic large area wound
Objective to explore the possibility of autologous hair follicle transplantation for the treatment of chronic large area wounds.
Methods chronic refractory wound area cases, patients informed consent, wound debridement, non purulent exudate control infection to fresh granulation tissue. After shaving the pillow, the British take knife blade thick skin, cut into 0.5cm * 0.5cm stamp; cut the skin area of hair follicle, under the microscope single from about 400 the root formation of epidermal hair follicles. The wound was divided into two zones: the planting area and blade thickness skin hair follicle area. Vaseline gauze, antibiotic saline gauze, limb braking. Change regularly, observe two wound healing; biopsy, HE staining, immunohistochemical detection of CK spectrum, vimentin (elastic protein) expression, observe the newborn skin.
The gross observation 2 weeks after operation: the hair follicle planting area can see pale epithelium grow by hair follicle transplantation outward, to transplant hair follicle center into skin island like pebbles distribution; blade thickness skin graft survival area, but slower growth. 8 weeks after operation: the hair follicle planting area of new skin connected into color consistent and growing part of the new sweat having slender hair; blade thick skin graft of skin scar, uneven color. 10 weeks after operation: the planting area of new skin hair follicles gradually mature, dark red, can withstand a certain degree of friction; split thickness skin grafting area scar obviously uneven. After April: hair follicle the planting area of newborn skin color, uniform texture, and fine sweat having hair growth; blade thickness skin area is obvious scar, poor texture. Immunohistochemistry of hair follicle planting area of 2 weeks by a long extension of new skin hair follicles, CK wide spectrum Up to high, and the emergence of spike structure between the epidermis and dermis (rete ridges), edge thickness skin areas have also seen a similar structure after.8 weeks, see a lot of new skin hair follicle growing area of spike like structures, and really epidermal dermoid tissue tightly, the expression of a large number of vimentin, sebaceous glands and sweat gland structure not found, edge skin graft has not been found. The gland structure zone
Conclusion: 1. After the autograft of autologous hair follicle, the epidermis cells take the hair follicle as the center to grow out and repair the wound, and the healing wound has a certain abrasion resistance.
(2) for the treatment of chronic large and refractory wounds, autologous hair follicle transplantation can be used as one of the options.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R329

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 劉菲;黃芩苷、人參莖葉皂苷對(duì)毛囊間充質(zhì)干細(xì)胞的影響[D];吉林農(nóng)業(yè)大學(xué);2012年

本文編號(hào)：1765477