【作者】 周乃慧；

【导师】 范卫新；

【作者基本信息】 南京医科大学 ， 皮肤病与性病学， 2008， 博士

【摘要】 研究背景毛囊是一种结构复杂的皮肤附属器官,由属于上皮成分的内毛根鞘、外毛根鞘、毛母质和属于真皮成分的真皮鞘、毛乳头组成。毛囊最为显著的特点是始终处于生长期、退行期和休止期的周期性循环中,许多激素、细胞因子、生长因子及其受体等相互作用,形成网络,共同调控着毛囊的周期性循环和毛发生长。研究证实,毛囊周围的血管形成具有毛发循环依赖性的特征,表现为生长期毛囊周围的血管密度和体积最大,到退行期和休止期逐渐减小。而且,毛囊周围的血管体积增加后,毛球的最大直径和毛干的直径也相应增加;相反,抑制毛囊周围的血管形成后,毛囊延迟进入生长期。因此,毛囊周围的血管形成在调节毛囊的周期性循环和毛发生长中起着十分重要的作用。毛囊周围的血管形成是一个非常复杂的过程,涉及众多的血管生成因子、血管活性因子和抑制性因子等。血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)又称血管渗透因子(vascular permeability factor,VPF)或血管调理素(vasculotropin),是近年来发现的一种毛囊起源的血管生成因子,生长期毛囊的毛乳头细胞、真皮鞘细胞和外毛根鞘细胞均可以合成与分泌VEGF,而退行期和休止期毛囊中VEGF的表达明显减少或消失。目前认为,VEGF促进毛发生长的可能机制有:①促进毛乳头细胞的增殖和迁移;②诱导毛囊周围的血管形成;③通过增加血管内皮细胞的通透性而促进营养物质的跨内皮转运,增加毛囊的营养供应;④刺激蛋白酶的合成,利于毛囊的重新塑形。据此,VEGF作为一种血管生成因子,其促进毛发生长的机理并不仅仅局限于诱导毛囊周围的血管形成。血管生成素(angiogenin)是由123个氨基酸组成、分子量为14 kDa的分泌性蛋白,属于胰腺核糖核酸酶超家族。1985年由Fett等首次从培养的人结肠癌细胞株HT-29的无血清上清中分离得到,具有很强的促进血管生成的能力。已经发现,血管生成素在许多肿瘤如前列腺癌、恶性黑素瘤和肝癌中表达明显上调,而且其表达水平与肿瘤的侵袭性和血管形成显著相关。另外,正常人血清、卵泡液、睾丸和子宫内膜中也表达血管生成素。因此,血管生成素的广泛分布提示其不仅参与血管形成,而且可能具有多种生物学功能。然而,到目前为止,血管生成素是否在人毛囊中表达及其对毛发生长的可能作用国内外尚未见报道。目的探讨血管生成素在人毛囊不同部位中的表达,并进一步研究血管生成素促进毛发生长的作用及其可能的机制。方法第一部分:人毛囊分离及人毛乳头细胞和外毛根鞘细胞培养利用解剖法在显微镜下分离完整的人毛囊,两步酶法分离培养人毛乳头细胞和外毛根鞘细胞。第二部分:血管生成素在人毛囊中的表达RT-PCR检测血管生成素mRNA在人毛囊中的表达,免疫荧光检测血管生成素蛋白在人毛囊不同部位中的表达。同时,ELISA检测血管生成素在体外培养的人毛囊上清中的表达,结合人毛囊的生长曲线,比较培养不同时间血管生成素的表达与毛囊生长长度之间的关系。第三部分:血管生成素在人毛乳头细胞中的表达RT-PCR检测血管生成素mRNA在人毛乳头细胞中的表达,western blot和免疫荧光检测血管生成素蛋白在人毛乳头细胞中的表达,ELISA检测血管生成素蛋白在人毛乳头细胞上清中的表达。第四部分:血管生成素在小鼠皮肤不同毛发周期中的表达利用松香/石蜡拔毛法拔除C57BL/6小鼠背部毛发,诱导毛囊同步进入生长期,分别在拔毛后第0、1、3、5、8、12、17、19、20、25天颈椎脱臼法处死小鼠,背部相同部位平行于脊椎取皮肤组织,TRIZOL提取总RNA,半定量RT-PCR检测血管生成素mRNA在小鼠皮肤不同毛发周期中的表达。第五部分:血管生成素局部注射对小鼠皮肤血管形成和毛发周期的影响利用松香/石蜡拔毛法拔除C57BL/6小鼠背部毛发,诱导毛囊同步进入生长期,拔毛后第0天在小鼠背部皮内注射100μL 10ng/μL重组人血管生成素(1μg),连续7天,对照组注射100μL 0.1%BSA-PBS。第10天观察注射部位皮肤的颜色和血管形成,同时取皮肤组织HE染色后,测量皮肤厚度和生长期Ⅵ毛囊比例。第六部分:血管生成素对体外培养的人毛囊生长的影响在体外培养的人毛囊中,加入不同浓度的重组人血管生成素(0-200ng/mL),同时加或不加抗人血管生成素的中和性抗体,培养6天,测量毛囊的生长长度。第七部分:血管生成素对体外培养的人毛乳头细胞增殖的影响在体外培养的人毛乳头细胞中,加入不同浓度的重组人血管生成素(0-200ng/mL),加或不加抗人血管生成素的中和性抗体,48h后,MTT法检测细胞增殖,流式细胞仪检测细胞周期。同时,将血管生成素的真核表达载体pEGFP-ANG转染人毛乳头细胞,设立转染pEGFP-C2组和单纯转染液组作为对照,转染后培养48h,MTT法检测细胞增殖。第八部分:血管生成素对体外培养的人外毛根鞘细胞增殖的影响在体外培养的人外毛根鞘细胞中,加入不同浓度的重组人血管生成素(0-200ng/mL),加或不加抗人血管生成素的中和性抗体,48h后,MTT法检测细胞增殖。结果第一部分:人毛囊分离及人毛乳头细胞和外毛根鞘细胞培养成功分离并培养了原代的人毛乳头细胞和外毛根鞘细胞。第二部分:血管生成素在人毛囊中的表达RT-PCR检测发现人毛囊表达血管生成素mRNA,PCR产物大形?52bp,经基因测序,与Gene Bank公布的序列完全一致。免疫荧光显示人毛囊的毛乳头和真皮鞘表达血管生成素蛋白,而外毛根鞘、内毛根鞘和毛母质不表达血管生成素蛋白,提示血管生成素表达在毛囊的真皮部位。ELISA检测结果显示体外培养的人毛囊上清中表达血管生成素蛋白,在培养的第2个48h内(第2-4天)血管生成素的表达和毛囊的生长长度均达到高峰,说明血管生成素的表达与毛囊的生长速度之间可能存在一定的相关性。第三部分:血管生成素在人毛乳头细胞中的表达RT-PCR检测发现培养的人毛乳头细胞表达血管生成素mRNA,PCR产物大小为352bp,经基因测序,与Gene Bank公布的序列完全一致。Western blot显示人毛乳头细胞表达14 kDa的血管生成素蛋白。免疫荧光进一步证实血管生成素蛋白表达在人毛乳头细胞的细胞浆内,而不表达在细胞核内。毛乳头细胞培养48h后,ELISA检测发现培养上清中血管生成素蛋白的含量为85.499±3.153 pg/10~5细胞。第四部分:血管生成素在小鼠皮肤不同毛发周期中的表达利用松香/石蜡拔毛法拔除C57BL/6小鼠背部毛发,可成功诱导毛囊同步进入生长期,拔毛后小鼠背部皮肤颜色逐渐由粉红色变为灰色、黑色,最后又由黑色变为灰色和粉红色。半定量RT-PCR发现小鼠皮肤中表达血管生成素mRNA,PCR产物大小为265bp,经基因测序,其序列与Gene Bank公布的完全一致。通过与内参GAPDH对比,拔毛后小鼠皮肤组织中血管生成素mRNA的表达具有毛发循环依赖性的特征,表现为生长期早期逐渐增加,生长期中晚期迅速增加,到生长期Ⅵ(拔毛后12天)达到高峰,以后在退行期早期迅速下降,到退行期晚期又逐步恢复到先前休止期时的水平。第五部分:血管生成素局部注射对小鼠皮肤血管形成和毛发周期的影响拔毛后连续7天局部皮内注射血管生成素(总计7μg),到第10天时局部皮肤颜色为灰黑色,与对照组相比无明显差异。但在皮肤反面,可以清楚地观察到注射血管生成素的小鼠皮肤血管的分支明显增多,而且其并不局限在注射部位。组织学上,注射血管生成素的小鼠皮肤厚度和生长期Ⅵ毛囊的比例明显高于对照组(p＜0.05)。第六部分:血管生成素对体外培养的人毛囊生长的影响体外培养的人毛囊中加入不同浓度的重组人血管生成素,培养6天后,25-100ng/mL重组人血管生成素能够明显促进体外培养的人毛囊生长(p＜0.001),并且具有一定的剂量依赖性。而低于25ng/mL的重组人血管生成素对人毛囊生长无明显影响(p＞0.05)。加入抗人血管生成素的中和性抗体,能够完全抑制重组人血管生成素促进毛囊生长的作用。第七部分:血管生成素对体外培养的人毛乳头细胞增殖的影响MTT检测发现低于12.5ng/mL重组人血管生成素对毛乳头细胞的增殖没有影响(p＞0.05),而12.5-200ng/mL重组人血管生成素能够明显促进毛乳头细胞增殖(p＜0.001),其中以50ng/mL和100ng/mL重组人血管生成素促进毛乳头细胞增殖的作用最为显著(p＜0.001)。而且,加入抗人血管生成素的中和性抗体能够完全阻断重组人血管生成素促进毛乳头细胞增殖的作用。人毛乳头细胞转染pEGFP-ANG后48h,MTT检测结果显示转染pEGFP-ANG组的毛乳头细胞增殖活性明显高于转染pEGFP-C2组和单纯转染液组(p＜0.001),而转染pEGFP-C2组和单纯转染液组毛乳头细胞的增殖无明显差异(p＞0.05)。流式细胞仪检测显示,12.5-200ng/mL重组人血管生成素能够明显增加毛乳头细胞S期细胞比率和细胞增殖指数(proliferation index,PI)(p＜0.05)。S期细胞比率与血管生成素之间具有剂量依赖性关系,随着血管生成素浓度的增加,S期细胞比率逐渐增加,当血管生成素为200 ng/mL时S期细胞比率最大。与对照组相比,血管生成素作用后PI明显增加,以100ng/mL血管生成素作用最为显著(p＜0.05)。第八部分:血管生成素对体外培养的人外毛根鞘细胞增殖的影响体外培养的人外毛根鞘细胞加入不同浓度的重组人血管生成素,培养48h后,MTT检测结果发现3.13-200ng/mL重组人血管生成素能够明显促进外毛根鞘细胞增殖(p＜0.001),且以12.5ng/mL作用最为显著(p＜0.001)。低于3.13ng/mL的重组人血管生成素对外毛根鞘细胞增殖无明显影响(p＞0.05)。同时加入抗人血管生成素的中和性抗体,可以完全阻断由血管生成素引起的外毛根鞘细胞增殖。结论1.血管生成素是一种新的毛囊起源的血管生成因子,表达在人毛囊的毛乳头和真皮鞘,且毛囊上清中血管生成素的表达量与毛囊的生长速度呈正相关。2.体外培养的人毛乳头细胞也能够合成与分泌血管生成素,血管生成素表达在人毛乳头细胞的细胞浆内。3.血管生成素mRNA在小鼠皮肤中的表达具有毛发循环依赖性的特征,生长期晚期表达最高,到退行期和休止期逐渐减少。4.血管生成素具有促进毛发生长和调节毛囊周期性循环的作用,其作用机制可能为:(1).诱导局部皮肤的血管形成;(2).诱导毛囊进入生长期;(3).促进毛乳头细胞的增殖;(4).促进外毛根鞘细胞的增殖。更多还原

【Abstract】 BackgroundHair follicle（HF）is a highly complex appendage of the skin,which consists of the mesenchymal components including dermal papilla and dermal sheath and the epithelial components such as outer root sheath and inner root sheath.Each hair follicle undergoes a cycle of growth（anagen phase）,regression（catagen phase）and resting（telogen phase）.Numerous growth factors,cytokines,hormones and the corresponding receptors are involved in regulating the hair growth and hair cycle.It has been proved that the angiogenesis around the hair follicle is hair-cycle dependent,with significant vascularization observed during the anagen phase,and blood vessel degeneration and disappearance during the catagen phase.As such, enhanced vascularization of the hair follicle has been shown to promote hair growth and to increase the diameter of both hair follicles and hair shafts.In contrast,the inhibition of perifollicular angiogenesis can delay the hair follicle entering into anagen.Therefore,perifollicular vasculature plays an important role in regulating the hair cycle and hair growth.The regulation of perifollicular angiogenesis is complex and it involves multiple vasoactive and angiogenic factors.Vascular endothelial growth factor（VEGF）,also named as vascular permeability factor or vasculotropin,is recently identified to be a kind of angiogenic factor derived from the hair follicle.Previous investigations indicated that VEGF was highly expressed in the dermal papilla,dermal sheath and outer root sheath of the anagen hair follicles,while VEGF decreased rapidly during the catagen and telogen.It has been demonstrated that VEGF can promote the proliferation and migration of the dermal papilla cells（DPCs）,and can induce the angiogenesis around the hair follicle.In addition,VEGF can enhance the local nutrition supply by increasing the permeability of the endothelial cells.It is also found that VEGF can stimulate protease synthesis.Together,VEGF is an important stimulator for hair growth and its role is not confined to inducing the perifolliclar angiogenesis.Human angiogenin,a 14 kDa polypeptide,is composed of 123 amino acids and is found to be a member of the ribonuclease superfamily. Angiogenin was initially purified from serum-free media conditioned by growth of a human adenocarcinoma cell line HT-29 based on its ability to initiate vascularization in the chicken embryo chorioallantoic membrane. Angiogenin is also expressed in a number of other tumors,including prostatic carcinoma,malignant melanoma and hepatocellular,as well as in normal tissues such as human testis and endometrium.In addition, angiogenin is a component of normal human plasma and follicular fluid. Its widespread expression in human tissues suggests that angiogenin may have other roles in addition to promoting angiogenesis.However,up to date,the expression of angiogenin in human hair follicles and the role of angiogenin in hair growth have not been described previously.ObjectivesTo investigate the expression of angiogenin in human hair follicles and therefore determine the effects of aniogenin on hair growth in vivo and in vitro.MethodsPartⅠ:The isolation of human hair follicles and the culture of human dermal papilla cells（DPCs）and outer root sheath（ORS） kerationocytesIntact human hair follicles were dissected under the microscope,and the DPCs and ORS keratinocytes were isolated and cultured by the two-step enzymic methods.PartⅡ:The expression of angiogenin in human hair folliclesRT-PCR and immunofluorescence analyses were applied to detect angioenin mRNA and angiogenin protein expression respectively in human hair follicles.In addition,ELISA was used to determine the concertration of angigenin protein in the culture medium of the human hair follicle,which was then compared with the hair follicle growth. PartⅢ:The expression of angiogenin in human DPCsRT-PCR was used to investigate angiogenin mRNA expression in human DPCs.Western blot was applied to determine angiogenin protein expression,which was further confirmed by immunofluorescence analysis.Angiogenin in the conditioned medium of cultured DPCs was detected by ELISA.PartⅣ:The expression of angiogenin mRNA in depilation induced murine hair cycleAnagen was induced in the back skin of the mice in the telogen phase of the hair cycle by melted wax/rosin.At 0,1,3,5,8,12,17,19,20, 25 days after depilation,mice were sacrificed and their back skin was harvested perpendicular to the paravertebral line.Total RNA was extracted from the skin with TRIZOL Reagent,and semi-quantitative RT-PCR was performed to assess angiogenin mRNA expression in murine skin phased at different hair cycle.PartⅤ:The effects of angiogenin local injection on the skin angiogenesis and the hair cycle in vivoAnagen was induced in the back skin of mice in the telogen phase of the hair cycle by melted wax/rosin.At day 0 after depilation,mice were administered a dorsal intradermal injection of 1μg recombinant human angiogenin dissolved in 100μL PBS containing 0.1%BSA（0.1% BSA-PBS）one daily for 7 days（totally 7μg）,and then sacrificed on day 10.Control mice were injected with 100μL 0.1%BSA-PBS.The skin color of the injection site and the blood vessel of the reverse side were observed and photographed on day 10.The skin sample from the injection site was embedded for cryosection and then was stained with HE for further analyses of the skin thickness and the percentage of hair follicles in anagenⅥ.PartⅥ:The effects of aniogenin on human hair follicle growth in vitroHuman occipital hair follicles in the anagenⅣstage were isolated and cultured in vitro.Recombinant human angiogenin was then added to the cultures in the absence or the presence of 0.8μg/mL anti-human angiogenin antibody,for final concentrations in the range of 0-200ng/mL. At days 0 and 6,the length of each hair follicle was measured under the microscope,enabling calculation of the 6-day elongation rate.PartⅦ:The effects of angiogenin on the proliferation of human DPCs in vitroDPCs were seeded on 96-well plates and were then cultured in 150μl serum-free DMEM for 24 hours before adding recombinant human angiogenin.Recombinant human angiogenin（0-200ng/mL final）in the absence or the presence of 0.8μg/mL anti-human angiogenin antibody were added to the cultures and were then incubated for 48 hours.MTT assay was used to detect cell proliferation,and flow cytometry was applied to analyze cell cycle.In addition,pEGFP-ANG,pEGFP-C2 and the transfected medium containing neither pEGFP-ANG nor pEGFP-C2 were transfected into human DPCs respectively by Lipofectamin^TM2000. After 48h cultures,MTT was performed to quantitate cell prolifertation.PartⅧ:The effects of angiogenin on the proliferation of human ORS keratinocytesORS keratinocytes were seeded on 96-well plates and were then cultured in 150μl serum-free keratinocytes medium for 24 hours before adding recombinant human angiogenin.Recombinant human angiogenin （0-200 ng/mL final）in the absence or the presence of 0.8μg/mL anti-human angiogenin antibody were added to the cultures and were then incubated for 48 hours.Cell proliferation was quantified by MTT assay.ResultsPartⅠ:The isolation of human hair follicles and the culture of human dermal papilla cells（DPCs）and outer root sheath（ORS） kerationocytesHuman DPCs and ORS keratinocytes were successfully isolated and cultured.PartⅡ:The expression of angiogenin in human hair folliclesAngiogenin mRNA was found in human hair follicles by RT-PCR analysis.After amplification with angiogenin-specific primers,we detected the PCR product of approximately 352bp,which was then confirmed by gene sequencing.Importantly,in human anagenⅣhair follicle,the dermal papilla as well as connective tissue sheath could also express angiogenin,while follicular epithelium including hair matrix, inner and outer root sheath did not show angiogenin deposition.And,in hair follicle cultures,single cultured hair follicle can also secrete angiogenin.Angiogenin amounts and hair follicle growth length reach the maximum during the second two days,which both declined rapidly within the third two days.PartⅢ:The expression of angiogenin in human DPCsRT-PCR analysis showed that angiogenin mRNA was expressed in cultured DPCs.The PCR product,approximately 352bp,was demonstrated to be angiogenin by gene sequencing.Western blot analysis of human DPC lysates with an angiogenin-specific antibody revealed a single band of 14 kDa.Also,we detected high levels of angiogenin in the cytoplasm of cultured DPCs,but not in DPCs nuclei.After 48 hours incubation in serum-free DMEM,DPCs can secrete an average of 85.499±3.153 pg/10⁵ cells angiogenin in the culture media.PartⅣ:The expression of angiogenin mRNA in the depilation induced murine hair cycleWax/rosin can successfully induce homogeneous anagen development in C57BL/6 mice,which was accompanied by the alternation of the skin color range from pink and grey to black,and then from black and grey to pink finally.Transcripts for angiogenin（265bp） were found in the mouse skin samples of all hair cycle stages,which were also confirmed by gene sequencing.After normalized against GAPDH, angiogenin exhibited an expression pattern in the sense that show a peak in late anagen and decline subsequently.Aniogenin transcript increased slowly in the early anagen,then rose rapidly during the mid to late anagen, and reached the maximum at anagenⅥ,followed by a rapid decrease in the early catagen and finally returned to the initial（telogen）level by late catagen.PartⅤ:The effects of angiogenin local injection on the skin angiogenesis and the hair cycle in vivoAngiogenin or vehicle solution（0.1%BSA-PBS）was injected intradermally for seven consecutive days（total 7μg）,and the mice were sacrificed on day 10.Skin color of the angiogenin injected site did not show significant difference from that of vehicle solution injected site. However,in the reverse side of the resected skin sample,increased branches of blood vessels,which not completely confining to the injection site,could be clearly observed in the angiogenin injected mouse skin,suggesting that angiogenin administration can initiate skin vascularization.Histologically,when compared with the vehicle solution injected mice,the angiogenin injected mice showed a significant increase in skin thickness（p＜0.05）.By quantitative histomorphometry,the difference became obvious when the percentage of hair follicles in anagenⅥin the skin of angiogenin and vehicle solution injected mice was analyzed statistically（p＜0.05）.PartⅥ:The effects of aniogenin on human hair follicle growth in vitroWe found that angiogenin ranging from 25 to 200ng/mL significantly increased hair follicle elongation in a dose-dependent manner.Hair follicle growth following treatment with 25ng/mL angiogenin was significant as compared with that of vehicle-treated controls（p＜0.001）.In contrast,angiogenin less than 25ng/mL did not affect hair follicle growth. And,neutralizing antibody against angiogenin can completely block the stimulation effects of exogenously added angiogenin,suggesting that recombinant angiogenin can promote human hair follicle elongation in vitro.PartⅦ:The effects of angiogenin on the proliferation of human DPCs in vitroUsing the MTT assay,we found that recombinant human angiogenin stimulated the proliferation of human DPCs in vitro.Angiogenin less than 12.5ng/mL resulted in no change in DPCs proliferation,however, angiogenin ranging from 12.5-200ng/mL caused a significant increase in DPCs proliferation as compared with vehicle-treated controls（p＜0.001）, with the maximal proliferation observed with the doses of 50ng/mL and 100ng/mL.Importantly,in the presence of 0.8μg/mL anti-human angiogenin antibody,DPCs keratinocytes proliferation stimulated by exogenous angiogenin could be completely neutralized.Flow cytometry showed that 12.5-200ng/mL recombinant human angiogenin can significantly increase the S%and the cell proliferation index（p＜0.05）.In addition,transfection of pEGFP-ANG into DPCs can significantly promote cell proliferation as compared with the controls（P＜0.05）.PartⅧ:The effects of angiogenin on the proliferation of human ORS keratinocytesMTT assay showed that recombinant human angiogenin can stimulate the proliferation of human ORS keratinocytes in vitro. Angiogenin less than 3.13ng/mL had no effects on the ORS keratinocytes proliferation,but 3.13-200ng/mL angiogenin could significantly stimulate the proliferation of ORS keratinocytes（p＜0.001）,with the optimal mitogenic activity obtained at the concentration of 12.5ng/mL. Importantly,in the presence of 0.8μg/mL anti-human angiogenin antibody, ORS keratinocytes proliferation stimulated by exogenous angiogenin could be completely neutralized.Conclusions1.Angiogenin is a newly angiogenic factor derived from the hair follicle and it is located at the dermal papilla and dermal sheath.Further, angiogenin amount in the conditioned medium of the cultured hair follicle is related to the growth length of the hair follicle.2.Cultured human DPCs can also express and secrete angiogenin, and angiogenin is presnet at the cytoplasm.3.The expression of angiogenin mRNA is hair-cycle dependent during the murine hair cycle in vivo,which exhibits an expression pattern in the sense that show a peak in late anagen and decline subsequently.4.Angiogenin can stimulate hair growth and regulate the hair cycle through the following mechanisms:（1）.induce local vascularization;（2）.induce anagen development;（3）.promote DPCs proliferation;（4）.stimulate ORS keratinocytes proliferation.更多还原