雌激素在黄褐斑发病中的作用及黄褐斑的治疗研究
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摘要
皮肤上出现色素沉着或色斑极大影响面容,且大部分属于后天性色素沉着。常见的有黄褐斑、雀斑、日晒斑、老年斑、外伤后色素沉着、手术后色素沉着等。其中黄褐斑发病率高、发病机理复杂、治疗效果不佳、复发率高,成为皮肤美容领域研究的热点和难点。
引起色素沉着的原因很多,亦十分复杂。凡影响色素沉着过程中任何一个环节都可能导致色素沉着,该过程包括：①黑素细胞内黑素小体的装配与黑素合成；②黑素小体由核周向树突远端转移；③黑素小体传递至邻近角质形成细胞；④黑素小体在角质形成细胞内再分布、降解。大致可分为遗传因素、物理性因素(如紫外线、日光曝晒等)、化学性因素(如重金属砷、铋、银、汞等物质刺激)、内分泌因素(妊娠、垂体、甲状腺、肾上腺、性腺功能紊乱等致黄体酮、雌激素水平变化)、炎症性因素和代谢性因素等几大类。
雌激素是主要由卵泡和黄体产生的甾体类激素,主要作用是促进生殖系统的发育,刺激女性第二性征的出现,影响代谢机能。既往研究已表明雌激素与乳腺疾病、生殖系统疾病、心脑血管、骨关节疾病均有密切的关系。
临床上我们发现,妊娠、口服避孕药、慢性疾病、化妆品都可能导致皮肤色素沉着,典型表现如黄褐斑。妇女怀孕后,体内激素水平会发生变化,如血液中雌激素、孕激素或促黑色素细胞激素水平增高,可能促使黑色素细胞活性增加,黑色素增多。沉着的色素常常于妊娠早、中期间出现,并逐渐加重至足月。而且有些患者在分娩后,其黄褐斑会持续存在。长期口服避孕药的妇女,由于体内激素水平的变化,也会和妊娠一样会诱发黄褐斑。一般症状从服药后1-20个月开始,停药后可逐渐消退。某些慢性疾病如肝脏病、慢性酒精中毒、结核、内脏肿瘤、甲状腺疾病及一些自身免疫性疾病等,特别是女性生殖器官疾病和月经不调、痛经、子宫附件炎、不孕症等患者面部也常常出现黄褐斑。这可能与卵巢、脑垂体、甲状腺等内分泌失调、激素水平紊乱有关。以上几种发病诱因中都可能有雌激素的作用,但雌激素与色素沉着疾病之间的关系研究甚少。
研究目的：
1、研究黄褐斑皮肤的组织学改变及角蛋白10(CK10)的表达情况。研究黄褐斑的色素分布特点及其与正常皮肤的差异。
2、研究女性黄褐斑患者病损部位的色素特点和血清雌激素水平的变化。探讨黄褐斑患者病损部位和正常皮肤雌激素受体p(ERβ)的分布情况,了解雌激素p在黄褐斑发病中的作用。进行人色素斑皮肤的黑色素细胞体外培养,观察培养的黑色素细胞的形态与生长特性。观察不同浓度外源性雌激素(已烯雌酚)对体外培养面部皮肤黑色素细胞的增殖的影响。观察雌激素对培养的面部黑色素细胞的酪氨酸酶活性及色素合成的影响。
3、研究应用计算机色素分析软件评估面部色素性疾病及其治疗效果。探讨黄褐斑的综合治疗方法,提高黄褐斑的治疗效果。研究黄褐斑患者病损部位激光治疗前后色素分布特点,了解激光治疗后黄褐斑色素的代谢变化。
研究方法：
1、取黄褐斑患者眼袋术后外眦多余皮肤,行石蜡切片的HE染色、Fontana-Masson染色和CK10的免疫组化染色,观察此处皮肤的病理学改变和CK10的分布情况。应用黄褐斑患者行外切口眼袋整形手术切取的外眦部色斑皮肤和周围正常皮肤分别行HE和Fontana-Masson染色,观察黄褐斑病损部和周围非病损皮肤的色素分布情况。
2、采用安琪儿软件对25例黄褐斑患者的病损部位色素水平进行分析,并利用放免法测定血清雌二醇等性激素水平。应用免疫组化法观察黄褐斑患者色素病损部和正常皮肤的雌激素受体p的表达分布情况,并分析其表达强度与色素的严重程度的相关性。获取黄褐斑患者眼袋术后皮肤标本,0.25%中性蛋白酶消化获取表皮,0.25%胰酶+0.02%EDTA消化获取细胞悬液,加入M254/HMGS条件培养基,进行黑色素细胞纯化培养,应用倒置显微镜观察细胞形态,采用多巴染色和S-100蛋白免疫组化染色进行鉴定。进行面部皮肤黑色素细胞的原代及传代培养,将第3代黑色素细胞接种于96孔板,每孔5×103个细胞,24h后换液,加入含1×10-～1×10-8M不同浓度已烯雌酚的培养基,孵育72h后MTT法测定黑素细胞增殖情况。获取眼袋术后皮肤标本,进行黑色素细胞纯化培养并鉴定。将第二代细胞传代接种,24小时后换液,加入含雌激素的黑色素细胞培养基,48小时后分别进行酪氨酸酶活性和色素合成的检测,72小时后消化收集细胞通过透射电镜检测超微结构的变化。
3、治疗前及复诊时采集患者色素性疾病区域的数码照片,输入计算机,应用色素分析软件系统测量患者皮肤色素性疾病的皮损面积、红、绿、蓝色的灰度值、平均灰度值、最大及最小灰度；对600例黄褐斑患者采用外用药物治疗、输液治疗、口服药物治疗及激光、光子等综合措施治疗。采用色素分析软件对治疗效果进行评估；选取伴下睑皮肤轻度松弛的黄褐斑志愿者,采用半面对照研究,一侧面部先行激光治疗3次。最后1次激光治疗后1周～1月行外路法双侧眼袋整复术,两侧色斑皮肤分别行HE和Fontana-Masson染色,观察黄褐斑患者病损部色素颗粒的分布变化情况。
结果：
1、黄褐斑皮损处表皮变薄,基底层细胞周围可见明显色素颗粒,基底上层、毛囊上段、真皮层也有色素颗粒分布,真皮胶原紊乱、断裂,皮肤附属器数量较少。CK10主要分布在表皮的基底上层和皮脂腺细胞的胞浆和腺泡的外层。黄褐斑病损处色素主要分布在表皮基底层和钉状突,成伞状或者帽样包绕在细胞核周围,在毛囊上段、基底上层及真皮层也有色素分布,与非病损皮肤相比,基底层、基底上层色素均有明显增多。非病损皮肤色素较少,角质层增厚。
2、黄褐斑皮损多为黄褐色,平均灰度值为122.84±20.59,患者体内雌二醇(E2)、孕酮(P)、黄体生成素(LH)和卵泡刺激素(FSH)水平与无色斑组相比均明显升高,而睾酮(T)、泌乳素(PRL)与无色斑组相比无显著差异。ERβ在色素斑处表皮层、毛囊外根鞘的细胞核强表达,表皮从基底层向上表达逐渐减弱,在汗腺、皮脂腺、真皮成纤维细胞、血管内皮细胞也有表达。在表皮层中的表达强度与色素的严重程度正相关。成功培养了人色素斑皮肤的黑色素细胞,细胞呈双极或者多级树突样,多巴染色和S-100蛋白免疫组化染色结果阳性。在10-8～10-6M浓度范围内己烯雌酚对体外培养黑色素细胞增殖有促进作用,作用强度与药物浓度呈正相关,10-5M以上己烯雌酚则对黑色素细胞的生长产生抑制或者毒性作用。加入10-6M雌激素后黑色素细胞色素合成和酪氨酸酶活性明显增加,经统计学检验P<0.05。超微结构显示细胞中黑素小体数量和成熟度增加。
3、采用计算机色素分析软件系统,可以测量色素性疾病皮损的面积和平均灰度,面积减少、平均灰度值增加说明治疗有效。经过综合治疗,144例患者(占24%)色斑颜色基本消退,168例患者(占28%)色斑有明显改善,204例患者(占34%)色斑有一定改善,总有效率达86%。黄褐斑病损经激光治疗后色素颗粒分散变小,向基底上层迁移,脱落的角质层也含有色素颗粒,真皮层色素颗粒也明显增加。
结论：
1、黄褐斑病损皮肤各层黑色素含量增多,皮肤组织结构多有老化表现。CK10在基底上层表达增强,其表达增强可能与表皮细胞过度分化、皮肤老化有关。黄褐斑患者色素主要分布在表皮基底层和钉状突,表皮全层色素颗粒增加,以基底层增加最为明显。
2、雌激素水平的变化可能在黄褐斑的发病中具有重要作用。雌激素受体β存在于皮肤及大多数附属器,可能在皮肤老化及皮肤色素沉积的过程中发挥重要作用。采用黑色素细胞条件培养基可以成功培养人色素斑皮肤的黑色素细胞,可以用作进一步的体外研究。一定浓度的己烯雌酚能促进黑色素细胞的增殖,最佳浓度为10-6M。雌激素对黄褐斑的发病可能具有促进作用。一定浓度的雌激素可以提高酪氨酸酶的活性,促进黑色素细胞合成色素。
3、计算机色素分析软件系统可以对色素性疾病的皮损的治疗效果做出比较客观和科学的评估。根据黄褐斑患者的不同情况,选择不同治疗方法,经过综合治疗可以取得满意疗效。激光治疗可以加速黄褐斑色素颗粒的代谢。
Background
Pure white and tender skin is taken for fair for the Asian ethnic. Pigmentation or stains appearing on the skin greatly affect their appearance. There are many types of acquired pigmentation disorders, for example melasma, freckles, sunburn plaques, senile plaques, post-traumatic pigmentation, pigmentation after surgery and so on.
There are many reasons for pigmentation, and its etiological factor is very complicated. Any one affecting the course of pigmentation may lead to this disease. The process includes:①melanosome assembly and melanin synthesis in melanocytes;②melanosome transfer from nuclear surrounds to dendritic remote;③melanosome transferred to neighboring keratinocytes;④melanosome re-distribution and degradation in keratinocytes. The major reason could be divided into genetic factors, physical factors (such as ultraviolet light, sunlight exposure, etc.), chemical factors (heavy metals such as arsenic, bismuth, silver, mercury and other incentives), endocrine factors (pregnancy, pituitary, thyroid, adrenal gland, gonadal function disorders, such as change of progesterone and estrogen levels), inflammatory factors and metabolic factors, and so on.
Estrogen is a steroidal hormones mainly produced by the follicle and corpus luteum. The main role is to promote the development of the reproductive system, to stimulate the female secondary sexual characteristics, and affect metabolism. Studies show that estrogen is closely related with breast disease, reproductive system diseases, cardio-cerebral blood vessels, bone and joint diseases.
Clinically we found that pregnancy, oral contraceptives, chronic diseases, cosmetics may lead to skin pigmentation, a typical example such as melasma. The hormone levels'change such as the increase of estrogen, progesterone hormone and MSH level blood in vivo of pregnant women may result in the increase of activity of melanocytes, and increase melanin synthesis. The pigmentation often appears in the pregnancy early, middle period, and gradually become obvious at last. And after delivery melasma will continue its existence for some patients. Long term oral contraceptives for women, melasma will be induced like as the pregnancy due to the change of hormone levels in vivo. Generally pigmentation will appear after taking medication for 20 months and gradually disappear until discontinuation drug. Melasma often appear in some chronic diseases such as liver disease, chronic alcoholism, tuberculosis, visceral tumors, thyroid disease and some autoimmune diseases, in particular female genital diseases and irregular menstruation, dysmenorrhea, uterine attachment inflammation, infertility. This may be related to the ovary, pituitary, thyroid and other endocrine hormone levels disorders. Estrogen may play an important role in the incidence of the above diseases, but the relationship between estrogen and pigmentation is rarely study.
In addition, melasma often appear in Asia, Latin America ethnic, and rarely occur in the white race. Therefore the role of estrogen in the pigmentation disease is rarely researched.
Human melanocytes mainly distribute in the human epidermis, dermis, hair follicle and eye choroid, etc. Melanocytes in the epidermal basal layer and hair follicles have the capacity of melanin secretion and synthesis. There are 100-2000 million melanocytes in the basal layer of the human epidermis, average about 1560 cells in per mm2. They distribute in the body surface symmetrically, but the depths vary due to different sites, generally more in head and skin folds, less in abdominal and back.
Maedak and other scholars have incubated normal human melanocytes with pituitary hormones, promoting melanoma cell hormones, FSH and ovarian hormones for estradiol (E2), estriol (E3), progesterone (P). After incubating for 2 days melanocytes became lager and took on dendritic-like appearances. They found in the trials that pituitary hormone can increase the activity of tyrosinase and tyrosinase-related protein-1, but ovarian hormone can only increase the activity of tyrosinase-related protein-1, without increasing the activity of tyrosinase in the same experimental conditions. This suggested that skin pigmentation caused by pituitary and ovarian hormones may be resulted by stimulating epidermal melanin generation in melanocytes, and E2 and P play an important role in the pathogenesis of melasma.
Some scholars have found estrogen and progesterone hormone levels in serum do not increase, sometimes even lower than normal in melasma patients. Especially melasma appears in some men. On the other hand pigment becomes deepen only in about 50% of pregnancy patients and oral contraceptives. So it is not the increase of sex hormone level which leads to melasma. Perhaps melanocytes in this part of patients is more sensitive to hormone, also perhaps there are differences in the melanoma cell-surface sex related hormone receptors of these patients.
Since the discovery of estrogen receptor (ER), reports on estrogen receptor is endless. Previous study on ER is limited to ERa. Until in 1996, a high affinity estrogen receptors ERβis isolated from the human and rat prostate tissues respectively. So researches on molecular biology of ER have entered a new stage particularly in the histological positioning of the two receptor subtypes and its relationship with gynecological endocrine. It is generally agreed that ER exists in multiple organs in vivo, and is closely related with menopausal and postmenopausal-related diseases. ERa is composited by 595 amino acids, which molecular weight is 66 KD and gene maps on chromosomes 6q25.1 zone, containing more than 140 kb of the base pair. ERβwas composited by 485 amino acids, which molecular weight of 54 KD, gene mapping on chromosome 14q22-24 areas, about 40 kb. ERa and ERβof human is very similar in the C domain and E domain, there is 97% and 59.1% homology. But there is only 17.5% and 17.9% homology in A/B domain and F domain. ERa and ERP exist in gender genital, heart, brain, bone, and kidney and so on widely. Combination with estrogen exert specific role in different organs.
Estrogen binding with cell surface estrogen receptor or other receptor activates the specific second messenger signal transduction system. When there is no estrogen, ER binds with co-inhibitor, and with no transcriptional activity. When estrogen in the tissue specific binding with ER, ER dissociates with co-inhibitor, phosphorylation occurs, dimers forms, and the co-activator factor is recruited, which activates transcription, then related proteins syntheses, and estrogen-target effects is exert. At present, it is considered there is two ways for ER to regulate the gene expression. First, ligand-dependent regulation, namely, the activated androgen receptor complex combined with specific estrogen response element (ERE) in DNA sequence, which directly activated the transcription; or binding with activated protein (API) transcription factor complex to control transcription indirectly. Second, non-ligand-dependent regulation, such as epidermal growth factor (EGF) can induce ERa phosphorylation and activate the transcription.
In short, the estrogen not only affects the skin hyperkeratosis, dermal fibrosis, melanin formation, but also regulates the function of skin appendages such as hair follicle, sweat glands, and sebaceous glands. So it is important in skin aging, pigment formation, hair growth, gland secretion, and skin cancer formation.The studies on the estrogen mainly concentrate on the role of the tumor, osteoporosis, and cardiovascular disease pathogenesis at present. As for estrogen and its receptor in the role of skin pigmentation have not been reported.
Objective
1、To investigate the histological changes of the skin with melasma and keratin 10 (CK10) expression. To study the melanin distribution in the melasma lesions and its differences with normal skin.
2、To investigate the pigment characteristics and the estrogen changes in serum of female melasma patients.To investigate the distribution of estrogen receptor (3(ERβ) in the skin of of melasma patients, to understand the role of estrogen in the pathogenesis of melasma. To investigate the culture method of melanocytes in melasma skin, and observe the morphology and biological properties of the cultured melanocytes in vitro. To observe the effect of different concentrations of exogenous estrogen(diethylstilbestrol)on cultured human melanocyte in face. And to observe the effect of estrogen on the tyrosinase activity and melanin synthesis of the cultured human facial skin melanocytes.
3、To investigate and evaluate the application of computer analysis software system on measurement of human pigmentation diseases. To investigate the ways to treat melasma with comprehensive methods, and to improve the treatment effectiveness of melasma. To investigate the melanin distribution before and after laser treatment in melasma patients in order to understand the melanin metabolism changes.
Methods
1、The excess skin in the lateral canthus was acquired after eyelid plasty operation for melasma patients, the paraffin sections was stained with HE, Fontana-Masson and CK10 immunohistochemical staining to observe the pathological changes of the melasma skin and the distribution of CK10. The excess skin in the lateral canthus was acquired during the blepharoplasty of melasma patients, the paraffin sections of which were stained with HE and Fontana-Masson to observe the melanin distribution in the melasma skin and normal skin.
2、The pigment characteristics were analyzed in 25 cases melasma patients with angel software system, serum estrogen level was detected using radioimmunoassay. The distribution of ERβwas detected by immunohistochemical stain in the pigmented lesion and normal skin in melasma patients, then the correlation of its expression intensity and severity of pigment was analyzed for comparison. The skin specimen was obtained from melasma patients after lower blepharoplasty. Epidermis was isolated by 0.25% dispase, and then single cell suspension was obtained after digested by 0.25% trypsin and 0.02%EDTA. M254/HMGS conditioned media was added to cultured the cells purifiedly, the cell morphology was observed through inverted microscope, S-100 protein immunohistochemical staining and dopa-staining was used for identification. The TRP-1, HMB-45 expression were studied by indirect immunofluorescence. The primary melanocytes were cultured from face skin. The 3rd generation subcultured melanocytes were seeded in 96 well plates, and each hole was added for 5×103 cells. The medium was exchanged after 24h, and various concentrations of diethylstilbestrol from 1×10-4～1×10-8M was added to medium. The melanocyte proliferation was determinated by MTT method after incubation for 72h. The excess skin in the lateral canthus was acquired during the blepharoplasty. The primary melanocytes were cultured and identified. The second generation cells were passaged and inoculated. The medium was changed after 24 hours, added with medium contained estrogen. After 48 hours, tyrosinase activity and pigment synthesis were detected, and the ultrastructural changes were detected after 72 hours.
3、Digital photos of pigment disorders in patients were collected before and after the treatment, and then were input the computer. The pigment analysis software system was applied to measure the area of lesions, and the red, green, blue gray value and the average gray of the facial pigment in patients, also include of the largest and the smallest gray.600 patients with melasma were treated with topical medications, infusion therapy, oral medications and lasers, intense pulsed light (IPL) and other comprehensive methods. To assess the effectiveness before and after treatment using pigment analysis software. Melasma patients whose lower eyelid skin was loosening were chosen. A split face study was carried on. One side of the face was treated with laser firstly for 3 times. lweek-l month after the last laser treatment, bilateral lower blepharoplasty was carried out. The skin of both sides was stained by HE and Fontana-Masson respectively to observe the change of pigment granules distribution in melasma lesions before and after laser treatment.
Results
1、The epidermis was thin in the melasma skin. And the melanin can be seen clearly around the basal layer cells.It could be seen in the upper basal layer, the upper hair follicle and dermis. Dermal collagen was disorder, fracture, the number of skin appendages was less than the normal skin. CK10 mainly distributed in the upper stratum of basal epidermis and the most outer layer of sebaceous glands. The melanin mainly distributed in the basal layer and the rete ridge(nail-like protrusion) in the melasma lesions, which was liked umbrella or cap wrapping around the nucleus. A number of melanin also distributed in the upper section of the hair follicles, the upper stratum of the basement and the dermis. Melanin was significantly more compared with the non-pigmented skin. Less melanin could be found in non-pigmented skin, but the corneum was significantly thicker.
2、The lesion on melasma skin was mostly brown. The average gray value was 122.84±20.59, estradiol(E2)、progesterone(P)、luteinizing hormone(LH) and follicle stimulating hormone(FSH) in vivo is higher compared with normal control group, and testosterone(T), prolactin(PRL) have no significantly difference with normal. ERβexpressed strongly in the nucleus of the cell of epidermis and hair follicle's outer root sheath in pigmentation lesion, and weaken from basal layer of epidermis to superficial. It expressed also in sebaceous glands, sweat glands, dermis fibroblast and vessel endothelial cell. It is positive correlation between the expression ERP in the epidermis and the severity of pigment. The human melanocytes in melasma skin were successfully cultured, and the cells were multi-dendritic. S-100 protein immunohistochemical staining and Dopa staining were positive. The TRP-1 and HMB-45 were positive expression by indirect immunofluorescence.10-8-10-6 M diethylstilbestrol could promote the proliferation of cultured melanocytes, and the intensity was positively correlated with the diethylstilbestrol concentration.10-5 M or more clould inhibited the proliferation of melanocytes. The tyrosinase activity and melanin synthesis were increased significantly after 10"6M estrogen was added (P< 0.05). The melanin bodies showed an increase in melanocytes by transmission electron microscope.
3、With pigment analysis computer software system, the area and the average gray value of the pigmented disease can be measured. The area of lesions reduction and the average gray value increase indicated the therapy is effective. After combined treatments, the pigmentation faded in 144 patients (24%), improved significantly in 168 patients (28%), improved mildly in 204 patients (34%), and the total effective rate was about 86%. Melasma lesions pigment mainly surrounded the basal cells. Certain of melanin distributed in the upper stratum of the basement, some located in the dermis. The pigment granules scattered and became smaller after laser treatment, some of which migrate to the upper stratum. Some of pigment granules located in the cuticle. The pigment granules in dermis also increased significantly after laser treatment.
Conclusion
1、The content of melanin increases in melasma skin, and skin aging features appear in the histological structure. CK10 expresses strongly in the upper stratum of basal epidermis which maybe relate with the excessive differentiation of epidermal cells and the skin aging.Melasma melanin mainly distributes in the basal layer and the rete ridge, and is significantly more compared with that in the normal skin.
2、The changes of estrogen in vivo may play an important role in the pathogenesis of melasma. ERβexpresses in the skin and it's appendant. It may be playing an important role in the process of the skin aging and skin pigmentation. Melanocytes can be successfully cultured in melasma skin with conditioned media insisted of special ingredients, which can be used for further investigation. The concentration of diethylstilbestrol can promote the cultured melanocytes proliferation in melasma, and the best concentration is 10-6M. The estrogen may play a important role on the incidence of melasma. Certain concentration of estrogen can improve the activity of tyrosinase and promote synthesis of melanin in melanocytes.
3、Computer pigment analysis software systems can be applied to assess the pigmented lesions objectively and scientificly. Different treatments should be selected according to the condition of patients, and a satisfactory effectiveness could be achieved with combined treatments for melasma. Laser treatment can accelerate the metabolism of melasma pigment granules.
引起色素沉着的原因很多,亦十分复杂。凡影响色素沉着过程中任何一个环节都可能导致色素沉着,该过程包括：①黑素细胞内黑素小体的装配与黑素合成；②黑素小体由核周向树突远端转移；③黑素小体传递至邻近角质形成细胞；④黑素小体在角质形成细胞内再分布、降解。大致可分为遗传因素、物理性因素(如紫外线、日光曝晒等)、化学性因素(如重金属砷、铋、银、汞等物质刺激)、内分泌因素(妊娠、垂体、甲状腺、肾上腺、性腺功能紊乱等致黄体酮、雌激素水平变化)、炎症性因素和代谢性因素等几大类。
雌激素是主要由卵泡和黄体产生的甾体类激素,主要作用是促进生殖系统的发育,刺激女性第二性征的出现,影响代谢机能。既往研究已表明雌激素与乳腺疾病、生殖系统疾病、心脑血管、骨关节疾病均有密切的关系。
临床上我们发现,妊娠、口服避孕药、慢性疾病、化妆品都可能导致皮肤色素沉着,典型表现如黄褐斑。妇女怀孕后,体内激素水平会发生变化,如血液中雌激素、孕激素或促黑色素细胞激素水平增高,可能促使黑色素细胞活性增加,黑色素增多。沉着的色素常常于妊娠早、中期间出现,并逐渐加重至足月。而且有些患者在分娩后,其黄褐斑会持续存在。长期口服避孕药的妇女,由于体内激素水平的变化,也会和妊娠一样会诱发黄褐斑。一般症状从服药后1-20个月开始,停药后可逐渐消退。某些慢性疾病如肝脏病、慢性酒精中毒、结核、内脏肿瘤、甲状腺疾病及一些自身免疫性疾病等,特别是女性生殖器官疾病和月经不调、痛经、子宫附件炎、不孕症等患者面部也常常出现黄褐斑。这可能与卵巢、脑垂体、甲状腺等内分泌失调、激素水平紊乱有关。以上几种发病诱因中都可能有雌激素的作用,但雌激素与色素沉着疾病之间的关系研究甚少。
研究目的：
1、研究黄褐斑皮肤的组织学改变及角蛋白10(CK10)的表达情况。研究黄褐斑的色素分布特点及其与正常皮肤的差异。
2、研究女性黄褐斑患者病损部位的色素特点和血清雌激素水平的变化。探讨黄褐斑患者病损部位和正常皮肤雌激素受体p(ERβ)的分布情况,了解雌激素p在黄褐斑发病中的作用。进行人色素斑皮肤的黑色素细胞体外培养,观察培养的黑色素细胞的形态与生长特性。观察不同浓度外源性雌激素(已烯雌酚)对体外培养面部皮肤黑色素细胞的增殖的影响。观察雌激素对培养的面部黑色素细胞的酪氨酸酶活性及色素合成的影响。
3、研究应用计算机色素分析软件评估面部色素性疾病及其治疗效果。探讨黄褐斑的综合治疗方法,提高黄褐斑的治疗效果。研究黄褐斑患者病损部位激光治疗前后色素分布特点,了解激光治疗后黄褐斑色素的代谢变化。
研究方法：
1、取黄褐斑患者眼袋术后外眦多余皮肤,行石蜡切片的HE染色、Fontana-Masson染色和CK10的免疫组化染色,观察此处皮肤的病理学改变和CK10的分布情况。应用黄褐斑患者行外切口眼袋整形手术切取的外眦部色斑皮肤和周围正常皮肤分别行HE和Fontana-Masson染色,观察黄褐斑病损部和周围非病损皮肤的色素分布情况。
2、采用安琪儿软件对25例黄褐斑患者的病损部位色素水平进行分析,并利用放免法测定血清雌二醇等性激素水平。应用免疫组化法观察黄褐斑患者色素病损部和正常皮肤的雌激素受体p的表达分布情况,并分析其表达强度与色素的严重程度的相关性。获取黄褐斑患者眼袋术后皮肤标本,0.25%中性蛋白酶消化获取表皮,0.25%胰酶+0.02%EDTA消化获取细胞悬液,加入M254/HMGS条件培养基,进行黑色素细胞纯化培养,应用倒置显微镜观察细胞形态,采用多巴染色和S-100蛋白免疫组化染色进行鉴定。进行面部皮肤黑色素细胞的原代及传代培养,将第3代黑色素细胞接种于96孔板,每孔5×103个细胞,24h后换液,加入含1×10-～1×10-8M不同浓度已烯雌酚的培养基,孵育72h后MTT法测定黑素细胞增殖情况。获取眼袋术后皮肤标本,进行黑色素细胞纯化培养并鉴定。将第二代细胞传代接种,24小时后换液,加入含雌激素的黑色素细胞培养基,48小时后分别进行酪氨酸酶活性和色素合成的检测,72小时后消化收集细胞通过透射电镜检测超微结构的变化。
3、治疗前及复诊时采集患者色素性疾病区域的数码照片,输入计算机,应用色素分析软件系统测量患者皮肤色素性疾病的皮损面积、红、绿、蓝色的灰度值、平均灰度值、最大及最小灰度；对600例黄褐斑患者采用外用药物治疗、输液治疗、口服药物治疗及激光、光子等综合措施治疗。采用色素分析软件对治疗效果进行评估；选取伴下睑皮肤轻度松弛的黄褐斑志愿者,采用半面对照研究,一侧面部先行激光治疗3次。最后1次激光治疗后1周～1月行外路法双侧眼袋整复术,两侧色斑皮肤分别行HE和Fontana-Masson染色,观察黄褐斑患者病损部色素颗粒的分布变化情况。
结果：
1、黄褐斑皮损处表皮变薄,基底层细胞周围可见明显色素颗粒,基底上层、毛囊上段、真皮层也有色素颗粒分布,真皮胶原紊乱、断裂,皮肤附属器数量较少。CK10主要分布在表皮的基底上层和皮脂腺细胞的胞浆和腺泡的外层。黄褐斑病损处色素主要分布在表皮基底层和钉状突,成伞状或者帽样包绕在细胞核周围,在毛囊上段、基底上层及真皮层也有色素分布,与非病损皮肤相比,基底层、基底上层色素均有明显增多。非病损皮肤色素较少,角质层增厚。
2、黄褐斑皮损多为黄褐色,平均灰度值为122.84±20.59,患者体内雌二醇(E2)、孕酮(P)、黄体生成素(LH)和卵泡刺激素(FSH)水平与无色斑组相比均明显升高,而睾酮(T)、泌乳素(PRL)与无色斑组相比无显著差异。ERβ在色素斑处表皮层、毛囊外根鞘的细胞核强表达,表皮从基底层向上表达逐渐减弱,在汗腺、皮脂腺、真皮成纤维细胞、血管内皮细胞也有表达。在表皮层中的表达强度与色素的严重程度正相关。成功培养了人色素斑皮肤的黑色素细胞,细胞呈双极或者多级树突样,多巴染色和S-100蛋白免疫组化染色结果阳性。在10-8～10-6M浓度范围内己烯雌酚对体外培养黑色素细胞增殖有促进作用,作用强度与药物浓度呈正相关,10-5M以上己烯雌酚则对黑色素细胞的生长产生抑制或者毒性作用。加入10-6M雌激素后黑色素细胞色素合成和酪氨酸酶活性明显增加,经统计学检验P<0.05。超微结构显示细胞中黑素小体数量和成熟度增加。
3、采用计算机色素分析软件系统,可以测量色素性疾病皮损的面积和平均灰度,面积减少、平均灰度值增加说明治疗有效。经过综合治疗,144例患者(占24%)色斑颜色基本消退,168例患者(占28%)色斑有明显改善,204例患者(占34%)色斑有一定改善,总有效率达86%。黄褐斑病损经激光治疗后色素颗粒分散变小,向基底上层迁移,脱落的角质层也含有色素颗粒,真皮层色素颗粒也明显增加。
结论：
1、黄褐斑病损皮肤各层黑色素含量增多,皮肤组织结构多有老化表现。CK10在基底上层表达增强,其表达增强可能与表皮细胞过度分化、皮肤老化有关。黄褐斑患者色素主要分布在表皮基底层和钉状突,表皮全层色素颗粒增加,以基底层增加最为明显。
2、雌激素水平的变化可能在黄褐斑的发病中具有重要作用。雌激素受体β存在于皮肤及大多数附属器,可能在皮肤老化及皮肤色素沉积的过程中发挥重要作用。采用黑色素细胞条件培养基可以成功培养人色素斑皮肤的黑色素细胞,可以用作进一步的体外研究。一定浓度的己烯雌酚能促进黑色素细胞的增殖,最佳浓度为10-6M。雌激素对黄褐斑的发病可能具有促进作用。一定浓度的雌激素可以提高酪氨酸酶的活性,促进黑色素细胞合成色素。
3、计算机色素分析软件系统可以对色素性疾病的皮损的治疗效果做出比较客观和科学的评估。根据黄褐斑患者的不同情况,选择不同治疗方法,经过综合治疗可以取得满意疗效。激光治疗可以加速黄褐斑色素颗粒的代谢。
Background
Pure white and tender skin is taken for fair for the Asian ethnic. Pigmentation or stains appearing on the skin greatly affect their appearance. There are many types of acquired pigmentation disorders, for example melasma, freckles, sunburn plaques, senile plaques, post-traumatic pigmentation, pigmentation after surgery and so on.
There are many reasons for pigmentation, and its etiological factor is very complicated. Any one affecting the course of pigmentation may lead to this disease. The process includes:①melanosome assembly and melanin synthesis in melanocytes;②melanosome transfer from nuclear surrounds to dendritic remote;③melanosome transferred to neighboring keratinocytes;④melanosome re-distribution and degradation in keratinocytes. The major reason could be divided into genetic factors, physical factors (such as ultraviolet light, sunlight exposure, etc.), chemical factors (heavy metals such as arsenic, bismuth, silver, mercury and other incentives), endocrine factors (pregnancy, pituitary, thyroid, adrenal gland, gonadal function disorders, such as change of progesterone and estrogen levels), inflammatory factors and metabolic factors, and so on.
Estrogen is a steroidal hormones mainly produced by the follicle and corpus luteum. The main role is to promote the development of the reproductive system, to stimulate the female secondary sexual characteristics, and affect metabolism. Studies show that estrogen is closely related with breast disease, reproductive system diseases, cardio-cerebral blood vessels, bone and joint diseases.
Clinically we found that pregnancy, oral contraceptives, chronic diseases, cosmetics may lead to skin pigmentation, a typical example such as melasma. The hormone levels'change such as the increase of estrogen, progesterone hormone and MSH level blood in vivo of pregnant women may result in the increase of activity of melanocytes, and increase melanin synthesis. The pigmentation often appears in the pregnancy early, middle period, and gradually become obvious at last. And after delivery melasma will continue its existence for some patients. Long term oral contraceptives for women, melasma will be induced like as the pregnancy due to the change of hormone levels in vivo. Generally pigmentation will appear after taking medication for 20 months and gradually disappear until discontinuation drug. Melasma often appear in some chronic diseases such as liver disease, chronic alcoholism, tuberculosis, visceral tumors, thyroid disease and some autoimmune diseases, in particular female genital diseases and irregular menstruation, dysmenorrhea, uterine attachment inflammation, infertility. This may be related to the ovary, pituitary, thyroid and other endocrine hormone levels disorders. Estrogen may play an important role in the incidence of the above diseases, but the relationship between estrogen and pigmentation is rarely study.
In addition, melasma often appear in Asia, Latin America ethnic, and rarely occur in the white race. Therefore the role of estrogen in the pigmentation disease is rarely researched.
Human melanocytes mainly distribute in the human epidermis, dermis, hair follicle and eye choroid, etc. Melanocytes in the epidermal basal layer and hair follicles have the capacity of melanin secretion and synthesis. There are 100-2000 million melanocytes in the basal layer of the human epidermis, average about 1560 cells in per mm2. They distribute in the body surface symmetrically, but the depths vary due to different sites, generally more in head and skin folds, less in abdominal and back.
Maedak and other scholars have incubated normal human melanocytes with pituitary hormones, promoting melanoma cell hormones, FSH and ovarian hormones for estradiol (E2), estriol (E3), progesterone (P). After incubating for 2 days melanocytes became lager and took on dendritic-like appearances. They found in the trials that pituitary hormone can increase the activity of tyrosinase and tyrosinase-related protein-1, but ovarian hormone can only increase the activity of tyrosinase-related protein-1, without increasing the activity of tyrosinase in the same experimental conditions. This suggested that skin pigmentation caused by pituitary and ovarian hormones may be resulted by stimulating epidermal melanin generation in melanocytes, and E2 and P play an important role in the pathogenesis of melasma.
Some scholars have found estrogen and progesterone hormone levels in serum do not increase, sometimes even lower than normal in melasma patients. Especially melasma appears in some men. On the other hand pigment becomes deepen only in about 50% of pregnancy patients and oral contraceptives. So it is not the increase of sex hormone level which leads to melasma. Perhaps melanocytes in this part of patients is more sensitive to hormone, also perhaps there are differences in the melanoma cell-surface sex related hormone receptors of these patients.
Since the discovery of estrogen receptor (ER), reports on estrogen receptor is endless. Previous study on ER is limited to ERa. Until in 1996, a high affinity estrogen receptors ERβis isolated from the human and rat prostate tissues respectively. So researches on molecular biology of ER have entered a new stage particularly in the histological positioning of the two receptor subtypes and its relationship with gynecological endocrine. It is generally agreed that ER exists in multiple organs in vivo, and is closely related with menopausal and postmenopausal-related diseases. ERa is composited by 595 amino acids, which molecular weight is 66 KD and gene maps on chromosomes 6q25.1 zone, containing more than 140 kb of the base pair. ERβwas composited by 485 amino acids, which molecular weight of 54 KD, gene mapping on chromosome 14q22-24 areas, about 40 kb. ERa and ERβof human is very similar in the C domain and E domain, there is 97% and 59.1% homology. But there is only 17.5% and 17.9% homology in A/B domain and F domain. ERa and ERP exist in gender genital, heart, brain, bone, and kidney and so on widely. Combination with estrogen exert specific role in different organs.
Estrogen binding with cell surface estrogen receptor or other receptor activates the specific second messenger signal transduction system. When there is no estrogen, ER binds with co-inhibitor, and with no transcriptional activity. When estrogen in the tissue specific binding with ER, ER dissociates with co-inhibitor, phosphorylation occurs, dimers forms, and the co-activator factor is recruited, which activates transcription, then related proteins syntheses, and estrogen-target effects is exert. At present, it is considered there is two ways for ER to regulate the gene expression. First, ligand-dependent regulation, namely, the activated androgen receptor complex combined with specific estrogen response element (ERE) in DNA sequence, which directly activated the transcription; or binding with activated protein (API) transcription factor complex to control transcription indirectly. Second, non-ligand-dependent regulation, such as epidermal growth factor (EGF) can induce ERa phosphorylation and activate the transcription.
In short, the estrogen not only affects the skin hyperkeratosis, dermal fibrosis, melanin formation, but also regulates the function of skin appendages such as hair follicle, sweat glands, and sebaceous glands. So it is important in skin aging, pigment formation, hair growth, gland secretion, and skin cancer formation.The studies on the estrogen mainly concentrate on the role of the tumor, osteoporosis, and cardiovascular disease pathogenesis at present. As for estrogen and its receptor in the role of skin pigmentation have not been reported.
Objective
1、To investigate the histological changes of the skin with melasma and keratin 10 (CK10) expression. To study the melanin distribution in the melasma lesions and its differences with normal skin.
2、To investigate the pigment characteristics and the estrogen changes in serum of female melasma patients.To investigate the distribution of estrogen receptor (3(ERβ) in the skin of of melasma patients, to understand the role of estrogen in the pathogenesis of melasma. To investigate the culture method of melanocytes in melasma skin, and observe the morphology and biological properties of the cultured melanocytes in vitro. To observe the effect of different concentrations of exogenous estrogen(diethylstilbestrol)on cultured human melanocyte in face. And to observe the effect of estrogen on the tyrosinase activity and melanin synthesis of the cultured human facial skin melanocytes.
3、To investigate and evaluate the application of computer analysis software system on measurement of human pigmentation diseases. To investigate the ways to treat melasma with comprehensive methods, and to improve the treatment effectiveness of melasma. To investigate the melanin distribution before and after laser treatment in melasma patients in order to understand the melanin metabolism changes.
Methods
1、The excess skin in the lateral canthus was acquired after eyelid plasty operation for melasma patients, the paraffin sections was stained with HE, Fontana-Masson and CK10 immunohistochemical staining to observe the pathological changes of the melasma skin and the distribution of CK10. The excess skin in the lateral canthus was acquired during the blepharoplasty of melasma patients, the paraffin sections of which were stained with HE and Fontana-Masson to observe the melanin distribution in the melasma skin and normal skin.
2、The pigment characteristics were analyzed in 25 cases melasma patients with angel software system, serum estrogen level was detected using radioimmunoassay. The distribution of ERβwas detected by immunohistochemical stain in the pigmented lesion and normal skin in melasma patients, then the correlation of its expression intensity and severity of pigment was analyzed for comparison. The skin specimen was obtained from melasma patients after lower blepharoplasty. Epidermis was isolated by 0.25% dispase, and then single cell suspension was obtained after digested by 0.25% trypsin and 0.02%EDTA. M254/HMGS conditioned media was added to cultured the cells purifiedly, the cell morphology was observed through inverted microscope, S-100 protein immunohistochemical staining and dopa-staining was used for identification. The TRP-1, HMB-45 expression were studied by indirect immunofluorescence. The primary melanocytes were cultured from face skin. The 3rd generation subcultured melanocytes were seeded in 96 well plates, and each hole was added for 5×103 cells. The medium was exchanged after 24h, and various concentrations of diethylstilbestrol from 1×10-4～1×10-8M was added to medium. The melanocyte proliferation was determinated by MTT method after incubation for 72h. The excess skin in the lateral canthus was acquired during the blepharoplasty. The primary melanocytes were cultured and identified. The second generation cells were passaged and inoculated. The medium was changed after 24 hours, added with medium contained estrogen. After 48 hours, tyrosinase activity and pigment synthesis were detected, and the ultrastructural changes were detected after 72 hours.
3、Digital photos of pigment disorders in patients were collected before and after the treatment, and then were input the computer. The pigment analysis software system was applied to measure the area of lesions, and the red, green, blue gray value and the average gray of the facial pigment in patients, also include of the largest and the smallest gray.600 patients with melasma were treated with topical medications, infusion therapy, oral medications and lasers, intense pulsed light (IPL) and other comprehensive methods. To assess the effectiveness before and after treatment using pigment analysis software. Melasma patients whose lower eyelid skin was loosening were chosen. A split face study was carried on. One side of the face was treated with laser firstly for 3 times. lweek-l month after the last laser treatment, bilateral lower blepharoplasty was carried out. The skin of both sides was stained by HE and Fontana-Masson respectively to observe the change of pigment granules distribution in melasma lesions before and after laser treatment.
Results
1、The epidermis was thin in the melasma skin. And the melanin can be seen clearly around the basal layer cells.It could be seen in the upper basal layer, the upper hair follicle and dermis. Dermal collagen was disorder, fracture, the number of skin appendages was less than the normal skin. CK10 mainly distributed in the upper stratum of basal epidermis and the most outer layer of sebaceous glands. The melanin mainly distributed in the basal layer and the rete ridge(nail-like protrusion) in the melasma lesions, which was liked umbrella or cap wrapping around the nucleus. A number of melanin also distributed in the upper section of the hair follicles, the upper stratum of the basement and the dermis. Melanin was significantly more compared with the non-pigmented skin. Less melanin could be found in non-pigmented skin, but the corneum was significantly thicker.
2、The lesion on melasma skin was mostly brown. The average gray value was 122.84±20.59, estradiol(E2)、progesterone(P)、luteinizing hormone(LH) and follicle stimulating hormone(FSH) in vivo is higher compared with normal control group, and testosterone(T), prolactin(PRL) have no significantly difference with normal. ERβexpressed strongly in the nucleus of the cell of epidermis and hair follicle's outer root sheath in pigmentation lesion, and weaken from basal layer of epidermis to superficial. It expressed also in sebaceous glands, sweat glands, dermis fibroblast and vessel endothelial cell. It is positive correlation between the expression ERP in the epidermis and the severity of pigment. The human melanocytes in melasma skin were successfully cultured, and the cells were multi-dendritic. S-100 protein immunohistochemical staining and Dopa staining were positive. The TRP-1 and HMB-45 were positive expression by indirect immunofluorescence.10-8-10-6 M diethylstilbestrol could promote the proliferation of cultured melanocytes, and the intensity was positively correlated with the diethylstilbestrol concentration.10-5 M or more clould inhibited the proliferation of melanocytes. The tyrosinase activity and melanin synthesis were increased significantly after 10"6M estrogen was added (P< 0.05). The melanin bodies showed an increase in melanocytes by transmission electron microscope.
3、With pigment analysis computer software system, the area and the average gray value of the pigmented disease can be measured. The area of lesions reduction and the average gray value increase indicated the therapy is effective. After combined treatments, the pigmentation faded in 144 patients (24%), improved significantly in 168 patients (28%), improved mildly in 204 patients (34%), and the total effective rate was about 86%. Melasma lesions pigment mainly surrounded the basal cells. Certain of melanin distributed in the upper stratum of the basement, some located in the dermis. The pigment granules scattered and became smaller after laser treatment, some of which migrate to the upper stratum. Some of pigment granules located in the cuticle. The pigment granules in dermis also increased significantly after laser treatment.
Conclusion
1、The content of melanin increases in melasma skin, and skin aging features appear in the histological structure. CK10 expresses strongly in the upper stratum of basal epidermis which maybe relate with the excessive differentiation of epidermal cells and the skin aging.Melasma melanin mainly distributes in the basal layer and the rete ridge, and is significantly more compared with that in the normal skin.
2、The changes of estrogen in vivo may play an important role in the pathogenesis of melasma. ERβexpresses in the skin and it's appendant. It may be playing an important role in the process of the skin aging and skin pigmentation. Melanocytes can be successfully cultured in melasma skin with conditioned media insisted of special ingredients, which can be used for further investigation. The concentration of diethylstilbestrol can promote the cultured melanocytes proliferation in melasma, and the best concentration is 10-6M. The estrogen may play a important role on the incidence of melasma. Certain concentration of estrogen can improve the activity of tyrosinase and promote synthesis of melanin in melanocytes.
3、Computer pigment analysis software systems can be applied to assess the pigmented lesions objectively and scientificly. Different treatments should be selected according to the condition of patients, and a satisfactory effectiveness could be achieved with combined treatments for melasma. Laser treatment can accelerate the metabolism of melasma pigment granules.
引文
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