毛囊干细胞向上皮细胞分化及其相关调控信号的初步研究

发布时间：2018-04-17 22:03

本文选题：毛囊干细胞 + Notch信号　；参考：《复旦大学》2007年博士论文

【摘要】： 第一部分小鼠鼠须及人头皮毛囊干细胞的定位及特点目的明确小鼠鼠须垫毛囊及成年男性额部毛囊干细胞的定位及分布特点，筛选鼠须垫切片快速高效的方法，比较正常成年男性与脂溢性脱发成年男性额部毛囊干细胞数量差别。方法取8周龄BALB／C小鼠双侧鼠须垫，自制标本固定器脱水，包埋切片，作常规HE染色及角蛋白15(CK15)免疫组化；取正常成年男性及脂溢性脱发成年男性额部头皮，脱水包埋切片，并作常规HE染色及角蛋白15免疫组化，显微镜下分析结果并比较。结果在显微镜下：①HE染色及CK15免疫组化能明确显示小鼠(BALB／C)鼠须垫毛囊毛囊外根鞘(ORS)、内跟鞘(IRS)、毛乳头(FP)及毛干(HS)及立毛肌等结构。在毛囊立毛肌止点外根鞘隆起的部位小范围角蛋白CK15表达明显。②HE染色及CK15免疫组化能明确显示成年男性额部头皮毛囊外根鞘、内跟鞘、毛乳头、皮脂腺、毛干及立毛肌等结构。但与小鼠不同，成年男性额部头皮毛囊CK15表达自皮脂腺开口水平以下的外根鞘狭长区域延展至中部，逐渐变淡。额部毛囊干细胞数量正常成年男性高于脂溢性脱发成年男性(P＜0.05)。结论小鼠鼠须毛囊干细胞位于毛囊立毛肌止点附近的隆突内，人头皮毛囊干细胞位于毛囊皮脂腺开口水平以下狭长区域的外根鞘内，并且额部毛囊干细胞数量正常成年男性高于脂溢性脱发成年男性。第二部分小鼠鼠须毛囊干细胞向短暂增殖细胞(TA细胞)分化的研究目的研究小鼠鼠须毛囊干细胞在外源性表皮生长因子(EGF)及创伤的诱导下，向TA细胞分化的情况。方法将8周龄BALB／C小鼠随机分4组，双侧鼠须垫去表皮，每天经口内颊粘膜注射EGF，分别为OIU，1000IU，5000IU，10000IU，观察创面恢复情况，确定最佳干预剂量。将8周龄BALB／C小鼠随机分三组，分别记为EGF组、创伤组和空白组。EGF干预采用每天经口内颊粘膜注射法(剂量5000IU)，创伤采用鼠须垫去表皮法。①分别于D0、D1、D3、D5、D7、D9(D-天)收集鼠须垫标本，脱水固定，做CK15及CK14免疫组化，CK15及CK14阳性细胞计数，方差分析不同组别不同时间有无差别。②取三组D0、D1、D3、D5、D7、D9小鼠鼠须垫，提取其毛囊真皮鞘细胞，FACS分析其CK15及CK14阳性细胞比率变化。结果三种剂量EGF均加速伤口愈合，OIUEGF组创面愈合时间为9天，1000IUEGF组为8天，5000IUEGF及10000IU组没有差别，均为7天。确定每天一次给予5000IU EGF为后续实验干预剂量。免疫组化结果：①CK15阳性细胞聚集于隆突部，与第一部分实验结果一致：a同组别不同时点比较，EGF组在不同时点CK15阳性细胞数目差别显著。第1天比第0天明显减少，第3天比第1天明显减少，第5天比第3天明显减少，第7天比第5天明显减少，第9天比第7天明显减少(均为P＜0.05)；创伤组在不同时间点CK15阳性细胞数目也呈现类似的变化，CK15阳性细胞渐次减少(均为P＜0.05)；空白组在不同时间CK15阳性细胞数目无明显变化(P＞0.05)。b同时点不同组别比较，在第0天时各组CK15阳性细胞数目无显著差别(P＞0.05)，在第1天，EGF组及创伤组与空白组都有明显差别(P＜0.05)，但两者之间无显著差别(P＞0.05)。第3，5，7，9天不同组别CK15阳性细胞数目差别显著，均为第空白组最多，其次为创伤组，EGF组组最少(均为P＜0.05)。②CK14阳性细胞分布范围则明显广于CK15阳性细胞，毛囊外根鞘及皮下组织均可见广泛表达：a同组别不同时点比较，EGF组在不同时点CK14阳性细胞数目差别显著。第1天比第0天明显增多，第1到7天缓慢增多，第9天比第7天又明显增多(均为P＜0.05)；创伤组在不同时间CK14阳性细胞细胞数目也不同，增多速度比较平缓；空白组在不同时间CK14阳性细胞数目无明显变化(P＞0.05)。b同时点不同组别比较，在第0天时各组CK14阳性细胞数目无显著差别(P＞0.05)，第1，3，5，7，9天不同组别CK14阳性细胞数目差别显著，均为空白组最少，其次为创伤组，EGF组最多(均为P＜0.05)。流式细胞分析结果：分析显示EGF组、创伤组CK15阳性细胞比率从第1天开始就明显下降，且下降速率前者大于后者，空白组没有明显变化。EGF组、创伤组CK14阳性细胞比率逐渐增加，且前者大于后者，空白组没有明显变化。结论①外源性EGF能加速小鼠创面的愈合，每天给予5000IU能显著加速创面的愈合。 ②EGF与创伤均能启动小鼠鼠须毛囊干细胞向TA细胞分化。EGF能加速创面的愈合，这种作用在干预的前期效果明显，随着时间的推移，作用减弱。第三部分EGF干预下小鼠鼠须毛囊外根鞘Notch信号变化的研究目的研究在外源性EGF及创伤的刺激下，小鼠毛囊外根鞘细胞中Notch信号的相关组分变化情况及信号的活化情况。方法将8周龄BALB／C小鼠均分三组，分别为EGF组、创伤组和空白组。EGF干预采用每天间断经口内颊粘膜注射法，创伤采用鼠须垫去表皮法。分别于D0、D1、D3、D5(D-天)收集鼠须垫标本，分离毛囊外根鞘，制成单细胞悬液，抽取mRNA，逆转录成cDNA，RT-PCR分别做Notchl、Delta、EGFR、及Notch信号的活化成分ICN基因测试，并比较各组有无差别。结果①各组细胞均能检测到Notchl、Delta、EGFR基因，且条带半定量分析基本相同，无明显区别(P＞0.05)。②EGF干预组在第1天能检测到ICN条带，灰度较淡。第1，3，5天均未测得。另外两组标本均未能测得ICN条带。结论①在外源性EGF刺激下，小鼠鼠须毛囊外根鞘细胞Notch信号被激活，且早期明显。提示EGF通过Notch信号途径促进创面的愈合。 ②创伤刺激未能激活Notch信号。第四部分人自体毛囊移植治疗慢性大面积创面的研究目的探索用自体毛囊移植治疗慢性大面积创面的可能性。方法选择慢性难治性大面积创面病例，患者知情同意，创面清创，控制感染至新鲜肉芽组织无脓性渗液。枕后备皮，英国刀取刃厚皮，修剪成0.5cm×0.5cm邮票状；切取取皮区毛囊，显微镜下单根分离约400根，形成去表皮毛囊。创面纵行分为两区：毛囊种植区及刃厚植皮区。凡士林油纱布覆盖，抗生素生理盐水纱布包扎，患肢制动。定期换药，观察两区创面愈合情况；取活检，HE常规染色，免疫组化检测CK广谱、vimentin(弹性蛋白)表达，观察新生皮肤情况。结果①大体观察术后2周：毛囊种植区可以看到苍白色新生上皮由移植毛囊向外长出，，以移植毛囊为中心成鹅卵石皮岛样分布；刃厚植皮区植皮成活，但生长较慢。术后8周：毛囊种植区新生皮肤连接成片，色泽基本一致，并有部分新生汗毛样纤细毛发长出；刃厚植皮区瘢痕化明显，皮肤色泽不均。术后10周：毛囊种植区新生皮肤逐渐成熟，成暗红色，能耐受一定程度摩擦；刃厚植皮区瘢痕化明显，凹凸不平。术后4月：毛囊种植区新生皮肤成熟，颜色质地均匀，并有纤细汗毛样毛发生长；刃厚植皮区则瘢痕明显，质地较差。②免疫组化毛囊种植区2周始新生表皮由毛囊延伸长出，CK广谱表达较高，并出现表皮与真皮间的钉突结构(rete ridges)，刃厚植皮区也看到类似结构。8周后，毛囊种植区新生皮肤见到大量钉突样结构，表皮与其下真皮样组织结合紧密，表达大量vimentin，未发现皮脂腺及汗腺结构，刃厚植皮区也未发现腺体结构。结论①自体毛囊移植后表皮细胞以毛囊为中心向外长出，修复创面。愈合创面有一定耐磨擦能力。 ②对于慢性大面积难治性创面的治疗，自体毛囊移植可以做为选择的方法之一。
[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.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R329

【引证文献】