OPN和MMP-9与外阴病变的相关性研究及阴痒洗剂治疗外阴硬化性苔癣的研究
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摘要
第一部分OPN和MMP-9与外阴病变的相关性研究
研究背景及意义
女性外阴的疾病主要包括外阴癌、外阴鳞状上皮内瘤样变(vulvar intraepithelial neoplasia,VIN)和外阴上皮内非瘤样变(non-neoplastic epithelial disorders,NNED)。外阴上皮内非瘤样变包括外阴硬化性苔癣(vulvar lichen sclerosus,VLS)、外阴鳞状上皮细胞增生和其他皮肤病。外阴癌的病理类型中最多见的是外阴鳞状细胞癌(vulvar squamous cell carcinoma,VSCC),约占90%。VSCC是由不同分化程度的鳞状上皮细胞构成的浸润性癌,虽然发病率较低,占女性生殖系统恶性肿瘤的3%-5%,但是在75岁以上妇女中其发病率约占25%,与最常见的女性恶性肿瘤-宫颈癌的发病率相近,并且近年来有发病年龄下降和发病率增高的趋势。由于VSCC病变部位特殊,而且缺少早期诊断的特异性指标,VSCC往往不能被早期发现。目前对于该病的治疗首选手术切除,放疗及化疗只能起到辅助的作用。但是术后外阴结构的改变和并发症的出现常常严重影响了患者的生活质量。所以有关VSCC的发生、进展及预后等方面的研究工作就具有重要的临床意义。
VIN和VLS被认为是VSCC的癌前病变。VIN是一种发生于女性外阴的疾病,发病率低,临床表现为白色、灰褐色或红色斑丘疹或斑块,可单发或多发。组织学表现以基底层以上表皮细胞成熟紊乱、胞核异常为特点。按照有无人乳头瘤病毒感染将其分为寻常型和分化型二种,这二种VIN均被认为是癌前病变。前者与高危型HPV病毒感染有关,主要患者为年轻女性；而后者与HPV感染无关,与外阴的长期慢性炎症刺激有关,主要患者为老年女性,并可能是VLS向VSCC进展的一个很短暂的上皮病变过程,大多数VSCC由第二个途径发展而来。VLS是妇科常见的慢性炎症性疾病,是一种较难治疗、易复发的疾病。最常见的症状是顽固性外阴瘙痒,晚上瘙痒严重,甚至影响睡眠。有些患者表现为排尿困难、性交痛和外阴烧灼感,还有一部分患者可以无明显症状。临床特点为外阴皮肤萎缩、粗糙、弹性差、皮肤颜色变白或花白、皱缩、变脆,常伴有皲裂和破溃难愈。长期患病会引起一系列后遗症,包括外阴粘连形成瘢痕、阴道口狭窄以及普遍存在的性心理障碍,大约3%-5%的患者可能发展为VSCC,因此有人将其视为外阴癌前病变。
VIN和VLS的早期发现和治疗可以阻止其向VSCC的发展,从而改善病人的预后。VLS如何进展到VSCC及VLS与VIN的关系目前尚不明确。所以,寻找一种方法能尽早的预测或发现VSCC的发生发展,以便提前干预,阻止疾病的进展,显得尤为重要。
恶性肿瘤的形成和进展依赖于细胞信号转导分子如细胞外基质蛋白的调节作用,而骨桥蛋白(osteopontin, OPN)就是这样一种细胞外基质蛋白。OPN是一种高度磷酸化的分泌型糖蛋白,在多种组织和细胞中合成并分泌到体液,与整合素和CD44受体结合,通过各种信号传导通路,在许多生理和病理过程中发挥着重要的作用。近几年来,已有研究证实,在乳腺癌、肺癌、结肠癌和许多其它类型的肿瘤中,OPN与肿瘤的进展和转移密切相关,并认为OPN是肿瘤发生和转移的预后因子。进一步研究发现,OPN与基质金属蛋白酶-9(matrixmetallo proteinase-9,MMP-9)等分子相互作用,降解细胞外基质,共同调节肿瘤细胞的粘附、进展和侵袭,所以OPN和MMP-9在肿瘤的发生、侵袭和转移中的关键调控作用日益受到人们的重视。MMP-9是基质金属蛋白酶家族中分子量最大且最重要的蛋白酶,是细胞外基质降解过程中的重要酶类。MMP-9可由多种细胞分泌,比如造血干细胞、中性粒细胞、内皮细胞以及各种肿瘤细胞等。MMP-9的主要作用是降解基底膜和细胞外基质,维持细胞外基质的生理动态平衡过程。在肿瘤的浸润和转移过程中,肿瘤细胞可通过分泌MMP-9降解细胞外基质,破坏细胞基底膜,并通过细胞外基质的改建,促进肿瘤新生血管的形成,从而影响肿瘤病人的转归及其预后。
然而,迄今为止,OPN和MMP-9的表达以及与外阴癌前病变及外阴鳞癌的相关性,国内外尚未见相关的研究报道。
本研究将检测OPN和MMP-9在VSCC、VIN和VLS的表达水平,探讨OPN和MMP-9与患者临床病理分级及预后的相关性。
研究目的
研究OPN和MMP-9在VSCC、VIN、VLS和正常对照外阴皮肤中的表达及其与临床病理分级及VSCC预后的相关性。探讨OPN和MMP-9在VSCC的发生和发展过程中的可能作用,并为进一步研究可靠的VSCC的预后因子提供科学依据。
研究方法
本研究回顾性研究山东大学齐鲁医院病理科和皮肤科2002年-2007年存档的石蜡组织标本,包括VSCC25例、VIN21例、VLS21例,另取正常外阴皮肤13例,采用免疫组化和原位杂交的方法,检测OPN和MMP-9的表达。对VSCC患者进行随访。应用SPSS13.0软件建立数据库并进行统计分析。卡方检验分析OPN和MMP-9与患者临床病理分级之间的关系,Kaplan-Meier法绘制患者的生存曲线,Log-rank检验比较患者的生存差别。
结果
1.无论是用免疫组织化学方法检测蛋白的表达,还是用原位杂交方法检测基因的表达,OPN和MMP-9在VSCC、VIN、VLS及正常组之间的表达有显著性差异(P<0.05),并且VSCC与VIN、VIN与VLS、VLS与正常组,每两组之间比较有显著性差异(P<0.05)。OPN和MMP-9的表达随着病变病理分级恶性程度的增加而增加。
2.免疫组化结果发现：OPN在VSCC、VIN的部分细胞外基质中高表达；VSCC周围正常组织中MMP-9的表达高于正常对照组。
3.OPN和MMP-9的表达无论是在蛋白水平,还是在基因水平均与VSCC患者的复发无显著相关性。(P>0.05)
4.高表达OPN蛋白的VSCC患者其术后5年总生存率显著低于低表达OPN蛋白的患者(P=0.033)。
5.高表达MMP-9基因的VSCC患者其术后5年总生存率显著低于低表达MMP-9基因的患者(P=0.037)。
6.高表达OPN蛋白的vscc患者其术后5年疾病特异性生存率显著低于低表达OPN蛋白的患者(P=0.048)。
7.卡方检验比较发现蛋白水平OPN与MMP-9的表达显著相关。21例高表达OPN的vscc组织中,20例为高表达MMP-9；4例低表达OPN的vscc患者中,0例为高表达MMP-9,二者相比差异有显著统计学意义(P=0.000)。OPN与MMP-9之间相关系数r=0.658。
8.卡方检验比较发现基因水平OPN与MMP-9的表达显著相关。21例高表达OPN基因的vscc组织中,19例为高表达MMP-9；4例低表达OPN基因的vscc患者中,0例为高表达MMP-9,二者相比差异有显著统计学意义(P=0.001)。OPN基因与MMP-9之间相关系数r=0.613。
结论
1.OPN和MMP-9可能参与vscc的发生及VIN和VLS的恶性进展。
2.OPN蛋白的高表达与患者术后5年疾病特异性生存率及5年总生存率相关,推测高表达的OPN蛋白对于预测vscc患者的不良预后可能有一定的参考价值。
3.MMP-9的表达可能在肿瘤的起始阶段起到了至关重要的作用,而OPN的改变相对是晚期事件。
第二部分阴痒洗剂治疗外阴硬化性苔癣的研究
研究背景
外阴硬化性苔癣(vulvar lichen sclerosus,VLS),是妇科及皮肤科门诊的常见病,是女性外阴组织发生色素改变及变性的一组疾病,为慢性炎症性病变。女性在任何年龄均可发病,但是以中老年女性为多。本病最常见的症状是顽固性外阴瘙痒,并且入睡时瘙痒明显,常常因为奇痒难忍而影响睡眠。但是也有一部分患者无明显症状,而仅仅是外阴皮肤黏膜颜色的变浅。如果患者因症状不明显而延误治疗,或治疗效果欠佳,病变长期发展,会导致很多的后遗症,常见的有外阴阴蒂处形成瘢痕、阴道口粘连狭窄,患者常常伴有性心理障碍。无奈的是,只有活检组织学病理诊断是确诊的唯一金标准。目前,外阴硬化性苔癣的病变原因,发病机制都还不清楚,研究发现本病的发生可能与很多因素有关,包括：自身免疫因素、遗传因素、新陈代谢、感染因素、内分泌因素及局部因素等。
目前对VLS没有确切有效的治疗方法,多年前,因为VLS可以发展成外阴癌,作为一种癌前病变,主张手术切除,但是手术后所带来的外阴的瘢痕形成等并发症更加严重的影响了病人的生活质量。目前认为,虽然VLS是一种癌前病变,但是癌变率低,只有大约3%-5%的患者可能发展为外阴鳞状细胞癌,治疗方案的选择时要充分权衡利弊,主张首选保守治疗。虽然对外阴硬化性苔癣的临床治疗也进行了广泛的探索,但是直到现在,也没有发现一种确切有效的治疗药物,国际上认为局部应用糖皮质激素涂擦患处,虽然有一定的疗效,但是需要一天涂抹多次,使用起来很不方便,并且停药一段时间后还会发作,需要继续重复使用。用的时间长了还会引起外阴皮肤粘膜萎缩而加重病变,有的还有皮肤刺激症状。鉴于西药治疗VLS的诸多弊端,我们有必要将研究目光转向毒副作用少、标本兼治、不会产生药物依赖性的传统医药。我们在积山东大学齐鲁医院对本病四十余年的临床与实验研究的基础上,自拟阴痒洗剂治疗本病,以研究其临床疗效和作用机理。
研究目的
本研究旨在观察阴痒洗剂治疗外阴硬化性苔癣的临床疗效,并进一步探讨其作用机理,以期探索出一条治疗外阴硬化牲苔癣的有效途径,为阴痒洗剂的推广应用提供有力的临床与实验依据。
研究方法
外阴硬化性苔癣的临床诊断、中医辨证分型标准及疗效评定标准均参考《中医妇科学》、《妇产科学》和《中药新药临床研究指导原则》制定。确诊为外阴硬化性苔癣的116例患者入选本研究,随机分为两组,分别是阴痒洗剂组58例,丙酸氯倍他索软膏西药组58例；另取38名正常妇女的空腹血清和13名正常妇女的外阴皮肤做正常对照组。重点观察阴痒洗剂治疗组和西药对照组治疗前后的外阴瘙痒情况、外阴皮肤粘膜的病变范围和颜色变化情况,检测治疗前后血清T细胞亚群CD3+、CD4+、CD8+和免疫球蛋白IgG、IgA、IgM的变化,观察治疗前后局部组织骨桥蛋白的表达情况,并观察治疗前后的组织病理学变化。
结果
1.阴痒洗剂能有效改善外阴硬化性苔癣患者的临床症状和体征,各中医辨证分型间疗效未见明显差异。
2.阴痒洗剂治疗组总有效率82.76%,西药对照组总有效率41.38%,两组比较差异有统计学意义(P<0.05),治疗组疗效优于西药对照组。
3.治疗前患者的血清CD3+、CD4+下降,CD8+升高,CD4+/CD8+下降,与正常对照组比较,差异有统计学意义(P<0.05)。阴痒洗剂治疗组治疗后CD3+、CD4+升高,CD8+下降,CD4+/CD8+升高,与正常对照组比较无显著性差异(P>0.05)。西药对照组于治疗前后各项指标比较无显著性差异(P>0.05)。两组治疗后各项指标比较差异有统计学意义(P<0.05)。
4.治疗前患者血清IgG水平下降,与正常对照组比较,差异有统计学意义(P<0.05)。阴痒洗剂治疗组治疗后IgG升高,与正常对照组比较无显著性差异(P>0.05)。西药对照组于治疗前后IgG水平比较无显著性差异(P>0.05)。两组治疗后IgG水平比较差异有统计学意义(P<0.05)。
5.治疗前患者病变皮肤的OPN表达显著增高,与正常对照组比较差异有统计学意义(P<0.05)。阴痒洗剂治疗组治疗后OPN表达降低,与正常对照组比较无显著性差异(P>0.05)。西药对照组于治疗前后各项指标比较均无显著性差异(P>0.05)。
6.组织病理学发现,治疗组治疗后表皮角化过度减轻,棘细胞层数增多,上皮脚出现,基底层空泡变性消失或明显改善,黑素细胞增多,真皮乳头恢复,真皮水肿和胶原均质化带消失或明显改善,炎细胞浸润明显减少。
结论
1.阴痒洗剂可以缓解外阴硬化性苔癣的症状、体征,临床疗效满意。
2.免疫功能的改变可能与VLS的病变相关,阴痒洗剂的治疗作用可能与免疫调节有关。
3.0PN可能与阴痒洗剂的免疫调节治疗作用有关。
意义
本研究为治疗外阴硬化性苔癣提供了一种有效的中药-阴痒洗剂,并初步探讨了阴痒洗剂的可能作用机理和骨桥蛋白在阴痒洗剂治疗中的作用,从而为阴痒洗剂的进一步推广应用提供有力的临床与实验依据。
Part I Study on the relationship of osteopontin and matrix metalloproteinases-9in vulvar diseases
Background and significance
Vulvar diseases mainly include vulvar carcinoma, vulvar intraepithelial neoplasia (VIN) and vulvar non-neoplastic epithelial disorders (VNED). VNED includes vulvar lichen sclerosus (VLS), vulvar squamous cell hyperplasia and other dermatoses. Vulvar squamous cell carcinoma (VSCC), an invasive carcinoma, is constructed by squamous cells of different degrees of differentiation. Squamous cell carcinomas (SCCs), the most common vulvar carcinomas, account for about90%of all vulvar malignancies and3%-5%of all gynaecological carcinomas. However, over the age of75the incidence of vulvar carcinoma peaks to approximately25%, making it as common as cervical carcinoma. VSCC represents an important medical challenge worldwide whose incidence is increasing at an alarming rate in young females. The diagnosis of VSCC is often delayed and the clinical management is very difficult. Surgery is considered to be the cornerstone of treatment of VSCC, and radio-and/or chemotheraphy only play a limited role. However, postoperative complications of vulvar impact seriously on the patient's quality of life. Therefore, the research work on the progression and prognosis of VSCC is very important.
Vulvar intraepithelial neoplasia (VIN) and vulvar lichen sclerosus (VLS) are all regarded as precancerous lesions. VIN, less seen in the vulva, is a histological diagnosis based on loss of squamous epithelial maturation associated with enlarged, hyperchromatic, pleomorphic nuclei and increased, usually atypical mitoses. There are two types of VIN according to the newest separate criteria:the usual type and the differentiated type. These two types are all regarded as pre-malignant lesions. The former is related to high-risk human papillomavirus and primarily affects younger patients. The latter is not related to HPV. It occurs in older women and leads to mostly differentiated keratinizing squamous cell carcinoma. This type relates to chronic inflammatory vulvar conditions such as VLS and accounts for the majority of all VSCC. VLS, often seen in the department of gynecology, is a chronic inflammatory skin disease, runing a relapsing and remitting course, and the management is difficult. The common symptom is pruritus, micturition difficult, algopareunia and burning sensation. At least one third of patients may be asymptomatic. Typically, the lesions are white plaques and papules, often with areas of erythema, ecchymosis, hyperkeratosis, pallor, fissuring, telangiectasia, hyperpigmentation, bullae, excoriation, oedema and/or ulceration. The lingering effects, most often encountered in long-term cases of VLS, include scarring, narrowing of the introitus which can make intercourse impossible, as well as widespread psychosexual disorder. The risk of developing squamous cell carcinoma of the vulva approaches3%-5%in women with VLS, which therefore is regarded as a pre-malignant lesion.
Early detection and treatment of VIN and VLS may prevent development of VSCC and improve its prognosis. The oncogenesis of VLS to VSCC and its connection with VIN also remains to be elucidated. In order to prevent the progression of VSCC, it is important to find a way to predict the development of vulvar precancerous lesions.
Progress of malignant tumors rely on the regulation of the extracellular matrix protein, which helps regulate the transfer of genetic modification and physiological changes, and OPN is such an extracellular matrix protein. Osteopontin is a highly phosphorylated glycoprotein and has been shown to be synthesized in a variety of tissues and cells and secreted into body fluids. OPN stimulates various signal transduction pathways through binding to its receptors, such as integrins and CD44variants, and therefore plays an active role in many physiological and pathological processes. It has also been shown that increased OPN expression correlates with tumor progression and metastasis in breast cancer, lung cancer, colon cancer, and in others cancers. It has been shown that OPN enhances the abilities of mobility and chemical invasiveness of malignant tumor cells possibly through regulating the activities of MMP-9, which degradate extracellular matrix. MMP-9is the largest and most important proteases of the matrix metalloproteinase family and plays an important role in the process of extracellular matrix degradation. MMP-9has been shown to be secreted in a variety of cells, such as hematopoietic stem cells, neutrophils, endothelial cells and a variety of tumor cells. The main role of MMP-9is to degrade the basement membrane and extracellular matrix, maintaining physiological homeostasis of extracellular matrix process. It has been shown that MMP-9degradation of extracellular matrix, promoting tumor angiogenesis, thereby affecting the outcome and prognosis of cancer patients in the process of tumor invasion and metastasis.
However, to the best of our knowledge, the expression of OPN and MMP-9in VSCC, VIN and VLS has not yet been elucidated so far.
In this study, we detect the expression and clinical significance of OPN and MMP-9in VSCC, VIN and VLS by the immunohistochemical method and chromogenic in situ hybridization.
Objective
We aimed to observe the expression of OPN and MMP-9in VSCC, VIN, VLS, and control. We investigated the role of OPN and MMP-9in the development of vulvar diseases and canceration, and investigated the relationship of OPN and MMP-9to the clinicopathologic factors and prognosis. The clinical-pathological prognostic factors of VSCC were investigated.
Methods
For immunohistochemical evaluation and chromogenic in situ hybridization, archival formalin-fixed, paraffin-embedded vulvar specimens from2002to2007were retrieved from the Department of Pathology and the Department of Dermatology at the Qilu Hospital of Shandong University. Samples from25VSCC,21VIN,21VLS and13control were collected to detect the expression of OPN and MMP-9. Immunohistochemical staining for OPN and MMP-9were performed using the streptavidin-peroxidase method.We analyzed the clinical pathology data of the patients with VSCC. All statistical analyses were performed with SPSS13.0statistical software. The x2test was performed to examine the association between OPN, MMP-9and variable clinicopathologic factors. Kaplan-Meier method was used to calculate the survival curves, and log-rank test was used to compare the statistical significance of difference between the survivals of patient subgroups.
Results
1. OPN and MMP-9expression increase with the degree of malignancy of vulvar lesions by the immunohistochemical method and chromogenic in situ hybridization (P <0.05).
2. OPN staining was found in the extracellular matrix in VSCC and VIN by the immunohistochemical method. The expression of MMP-9was notable increase in normal epidermis adjacent to VSCC when compared with normal skin by the immunohistochemical method.
3. OPN overexpression and high MMP-9were no significantly associated with tumor relapse by the immunohistochemical method and chromogenic in situ hybridization (P<0.05).
4. Univariate survival analysis showed that patients with OPN overexpression had a significantly poor overall survival at5years after operation by the immunohistochemical method (P=0.033).
5. Univariate survival analysis showed that patients with MMP-9overexpression had a significantly poor overall survival at5years after operation by chromogenic in situ hybridization (P=0.037).
6. Univariate survival analysis showed that patients with OPN overexpression had a significantly poor disease-specific survival at5years after operation by the immunohistochemical method (P=0.048)
7. The x2test showed that OPN overexpression was significantly associated with high MMP-9by the immunohistochemical method (P=0.000).
8. The x2test showed that OPN overexpression was significantly associated with high MMP-9by chromogenic in situ hybridization (P=0.001).
Conclusion
1. OPN and MMP-9are involved in the development of the vulvar diseases and the malignant progression of VSCC.
2. OPN protein overexpression is significantly associated with poor survival and may have clinical potential as a promising predictor to identify individuals with poor prognostic potential.
3. MMP-9may represent an early stage of malignant transformation in VSCC, while OPN perhaps a late stage.
Part Ⅱ Study of Yinyang lotion in the treatment of vulvar lichen sclerosus
Background
Vulvar lichen sclerosus (VLS), often seen in the department of gynecology, is a chronic inflammatory skin disease, runs a relapsing and remitting course, and the management is difficult. VLS occurs at all ages, and has a bimodal peak incidence in menopausal women. The common symptom is pruritus, micturition difficult, burning sensation and algopareunia. At least one third of patients may be asymptomatic. Typically, the lesions are white plaques and papules, often with areas of erythema, ecchymosis, hyperkeratosis, pallor, fissuring, telangiectasia, hyperpigmentation, excoriation, oedema and/or ulceration. The lingering effects, most often encountered in long-term cases of VLS, include scarring, narrowing of the introitus which can make intercourse impossible, as well as widespread psychosexual disorder. The risk of developing squamous cell carcinoma of the vulva approaches3%-5%in women with VLS. The histological diagnosis is the only method to make a definite diagnosis. Up to now, the pathogenesis of VLS is incompletely understood. The autoimmunity, endocrine factor, enetic factor, metabolism, infection and local factor may be involved in it. The early diagnosis and treatment for VLS is a challenge for gynaecologists. In the past few years, scholars investigated the aetiology, pathogenesy and management of VLS, however, extensively approved management. The local corticosteroids is regarded as the first choice, but its side effects, including atrophia and skin stimulus, make the treatment to run into a predicament. The conbined treatment of traditional Chinese medicine and western medicine was used by internal scholars and got satisfactory results. We used Yinyang lotion to treat VLS on the basis of clinical and empirical study for more than40years in Qilu Hospital of Shandong University, and investigated its clinical curative effect and mechanism of action.
Objective
To observe the clinical curative effect of Yinyang lotion on VLS, and investigate the mechanism of action, so as to explore an effective way for the prevention and treatment of VLS, and provide strong theoretical and practical basis for the wide administration of Kebai cream.
Methods
We referred to the medicine teaching material edited by the government (Chinese Gynaecology, Obstetrics and Gynecology), Practical Obstetrics and Gynecology (second edition) and The Clinical Guidlines for the New Chinese Medicine,(Enacted by the Department of Health of China in1993and2002), and instituted the clinical diagnosis, the criterion of differentiation of symptoms and signs for classification of syndrome with traditional Chinese medicine and evaluation criterion of curative effect of VLS.
116patients with VLS were divided into case group and control group randomly. The case group (58patients) was treated with Yinyang lotion while the control group (58patients) was treated with clobetasol propionate ointment. The serum from38healthy women and vulvar tissue from13normal women were collected as normal control. The changes of the symptoms and signs such as pruritus, depigment and atrophy, were followed up during the period of the management. We detected the serum of the patients and the healthy women for T cell subsets (mainly CD3+, CD4+, CD8+T cells), IgG, IgA, IgM. The expression of osteopontin (OPN) and the histomorphology change in the prior and post-treatment were observed.
Results
1. The symptoms and signs of the case group, such as itching, depigment and atrophy, were greatly improved after treatment. And the validity among the types of case group was found no significant difference.
2. The total effective rate of case group is82.76%, while the control group is41.38%. By comparation, a statistical meaning can be educed (P<0.05), which is that the curative effect of the case group is better than the control group.
3. the expression of CD3+, CD4+and CD4+/CD8+T cells decrease significantly, while the significant increases of CD8+T cells were observed before the treatment (P <0.05). However, significant increases of CD3+, CD4+and CD4+/CD8+T cells, and the significant decreases of CD8+T cells were observed after the treatment with Yinyang lotion (P<0.05). There was no difference in results for T cells expression in the prior and post-treatment with clobetasol propionate ointment (P>0.05). After treatment, the significant difference in the expression of T cells between case group and control group was observed (P<0.05).
4. Significant decreases in the level of IgG were observed before treatment(P< 0.05). However, significant increases of IgG were detected after the treatment with Yinyang lotion (P<0.05). There was no difference in results for IgG in the prior and post-treatment with clobetasol propionate ointment (P>0.05). After treatment, the significant difference in level of IgG between case group and control group was observed (P<0.05).
5. By immunohistochemistry, significant decreases of OPN immunostaining cells were observed after the treatment with Yinyang lotion (P<0.05). There was no difference in results for OPN expression in the prior and post-treatment with clobetasol propionate ointment (P>0.05).
6. Observation by light microscope, after the treatment with Yinyang lotion, showed that lessened hyperkeratinization in the epidermis, more prickle cell layer, appearing rete ridges, improved obviously basal keratinocyte hydropic degeneration and vacuolization, melanocytosis, restored dermal papilla, obvious improvement or loss of edema and homogenization of collagen in the upper dermis, lessened obviously lymphocytic infiltration.
Conclusion
1. Yinyang lotion has a good clinical curative effect, and is an ideal medicine for theraphy of VLS.
2. Changes in immune function may be associated with VLS lesions, Yinyang lotion therapeutic effect may be related to immune regulation.
3. The mechanism of Yinyang lotion may adjust the immune function, OPN may be involved in this process.
研究背景及意义
女性外阴的疾病主要包括外阴癌、外阴鳞状上皮内瘤样变(vulvar intraepithelial neoplasia,VIN)和外阴上皮内非瘤样变(non-neoplastic epithelial disorders,NNED)。外阴上皮内非瘤样变包括外阴硬化性苔癣(vulvar lichen sclerosus,VLS)、外阴鳞状上皮细胞增生和其他皮肤病。外阴癌的病理类型中最多见的是外阴鳞状细胞癌(vulvar squamous cell carcinoma,VSCC),约占90%。VSCC是由不同分化程度的鳞状上皮细胞构成的浸润性癌,虽然发病率较低,占女性生殖系统恶性肿瘤的3%-5%,但是在75岁以上妇女中其发病率约占25%,与最常见的女性恶性肿瘤-宫颈癌的发病率相近,并且近年来有发病年龄下降和发病率增高的趋势。由于VSCC病变部位特殊,而且缺少早期诊断的特异性指标,VSCC往往不能被早期发现。目前对于该病的治疗首选手术切除,放疗及化疗只能起到辅助的作用。但是术后外阴结构的改变和并发症的出现常常严重影响了患者的生活质量。所以有关VSCC的发生、进展及预后等方面的研究工作就具有重要的临床意义。
VIN和VLS被认为是VSCC的癌前病变。VIN是一种发生于女性外阴的疾病,发病率低,临床表现为白色、灰褐色或红色斑丘疹或斑块,可单发或多发。组织学表现以基底层以上表皮细胞成熟紊乱、胞核异常为特点。按照有无人乳头瘤病毒感染将其分为寻常型和分化型二种,这二种VIN均被认为是癌前病变。前者与高危型HPV病毒感染有关,主要患者为年轻女性；而后者与HPV感染无关,与外阴的长期慢性炎症刺激有关,主要患者为老年女性,并可能是VLS向VSCC进展的一个很短暂的上皮病变过程,大多数VSCC由第二个途径发展而来。VLS是妇科常见的慢性炎症性疾病,是一种较难治疗、易复发的疾病。最常见的症状是顽固性外阴瘙痒,晚上瘙痒严重,甚至影响睡眠。有些患者表现为排尿困难、性交痛和外阴烧灼感,还有一部分患者可以无明显症状。临床特点为外阴皮肤萎缩、粗糙、弹性差、皮肤颜色变白或花白、皱缩、变脆,常伴有皲裂和破溃难愈。长期患病会引起一系列后遗症,包括外阴粘连形成瘢痕、阴道口狭窄以及普遍存在的性心理障碍,大约3%-5%的患者可能发展为VSCC,因此有人将其视为外阴癌前病变。
VIN和VLS的早期发现和治疗可以阻止其向VSCC的发展,从而改善病人的预后。VLS如何进展到VSCC及VLS与VIN的关系目前尚不明确。所以,寻找一种方法能尽早的预测或发现VSCC的发生发展,以便提前干预,阻止疾病的进展,显得尤为重要。
恶性肿瘤的形成和进展依赖于细胞信号转导分子如细胞外基质蛋白的调节作用,而骨桥蛋白(osteopontin, OPN)就是这样一种细胞外基质蛋白。OPN是一种高度磷酸化的分泌型糖蛋白,在多种组织和细胞中合成并分泌到体液,与整合素和CD44受体结合,通过各种信号传导通路,在许多生理和病理过程中发挥着重要的作用。近几年来,已有研究证实,在乳腺癌、肺癌、结肠癌和许多其它类型的肿瘤中,OPN与肿瘤的进展和转移密切相关,并认为OPN是肿瘤发生和转移的预后因子。进一步研究发现,OPN与基质金属蛋白酶-9(matrixmetallo proteinase-9,MMP-9)等分子相互作用,降解细胞外基质,共同调节肿瘤细胞的粘附、进展和侵袭,所以OPN和MMP-9在肿瘤的发生、侵袭和转移中的关键调控作用日益受到人们的重视。MMP-9是基质金属蛋白酶家族中分子量最大且最重要的蛋白酶,是细胞外基质降解过程中的重要酶类。MMP-9可由多种细胞分泌,比如造血干细胞、中性粒细胞、内皮细胞以及各种肿瘤细胞等。MMP-9的主要作用是降解基底膜和细胞外基质,维持细胞外基质的生理动态平衡过程。在肿瘤的浸润和转移过程中,肿瘤细胞可通过分泌MMP-9降解细胞外基质,破坏细胞基底膜,并通过细胞外基质的改建,促进肿瘤新生血管的形成,从而影响肿瘤病人的转归及其预后。
然而,迄今为止,OPN和MMP-9的表达以及与外阴癌前病变及外阴鳞癌的相关性,国内外尚未见相关的研究报道。
本研究将检测OPN和MMP-9在VSCC、VIN和VLS的表达水平,探讨OPN和MMP-9与患者临床病理分级及预后的相关性。
研究目的
研究OPN和MMP-9在VSCC、VIN、VLS和正常对照外阴皮肤中的表达及其与临床病理分级及VSCC预后的相关性。探讨OPN和MMP-9在VSCC的发生和发展过程中的可能作用,并为进一步研究可靠的VSCC的预后因子提供科学依据。
研究方法
本研究回顾性研究山东大学齐鲁医院病理科和皮肤科2002年-2007年存档的石蜡组织标本,包括VSCC25例、VIN21例、VLS21例,另取正常外阴皮肤13例,采用免疫组化和原位杂交的方法,检测OPN和MMP-9的表达。对VSCC患者进行随访。应用SPSS13.0软件建立数据库并进行统计分析。卡方检验分析OPN和MMP-9与患者临床病理分级之间的关系,Kaplan-Meier法绘制患者的生存曲线,Log-rank检验比较患者的生存差别。
结果
1.无论是用免疫组织化学方法检测蛋白的表达,还是用原位杂交方法检测基因的表达,OPN和MMP-9在VSCC、VIN、VLS及正常组之间的表达有显著性差异(P<0.05),并且VSCC与VIN、VIN与VLS、VLS与正常组,每两组之间比较有显著性差异(P<0.05)。OPN和MMP-9的表达随着病变病理分级恶性程度的增加而增加。
2.免疫组化结果发现：OPN在VSCC、VIN的部分细胞外基质中高表达；VSCC周围正常组织中MMP-9的表达高于正常对照组。
3.OPN和MMP-9的表达无论是在蛋白水平,还是在基因水平均与VSCC患者的复发无显著相关性。(P>0.05)
4.高表达OPN蛋白的VSCC患者其术后5年总生存率显著低于低表达OPN蛋白的患者(P=0.033)。
5.高表达MMP-9基因的VSCC患者其术后5年总生存率显著低于低表达MMP-9基因的患者(P=0.037)。
6.高表达OPN蛋白的vscc患者其术后5年疾病特异性生存率显著低于低表达OPN蛋白的患者(P=0.048)。
7.卡方检验比较发现蛋白水平OPN与MMP-9的表达显著相关。21例高表达OPN的vscc组织中,20例为高表达MMP-9；4例低表达OPN的vscc患者中,0例为高表达MMP-9,二者相比差异有显著统计学意义(P=0.000)。OPN与MMP-9之间相关系数r=0.658。
8.卡方检验比较发现基因水平OPN与MMP-9的表达显著相关。21例高表达OPN基因的vscc组织中,19例为高表达MMP-9；4例低表达OPN基因的vscc患者中,0例为高表达MMP-9,二者相比差异有显著统计学意义(P=0.001)。OPN基因与MMP-9之间相关系数r=0.613。
结论
1.OPN和MMP-9可能参与vscc的发生及VIN和VLS的恶性进展。
2.OPN蛋白的高表达与患者术后5年疾病特异性生存率及5年总生存率相关,推测高表达的OPN蛋白对于预测vscc患者的不良预后可能有一定的参考价值。
3.MMP-9的表达可能在肿瘤的起始阶段起到了至关重要的作用,而OPN的改变相对是晚期事件。
第二部分阴痒洗剂治疗外阴硬化性苔癣的研究
研究背景
外阴硬化性苔癣(vulvar lichen sclerosus,VLS),是妇科及皮肤科门诊的常见病,是女性外阴组织发生色素改变及变性的一组疾病,为慢性炎症性病变。女性在任何年龄均可发病,但是以中老年女性为多。本病最常见的症状是顽固性外阴瘙痒,并且入睡时瘙痒明显,常常因为奇痒难忍而影响睡眠。但是也有一部分患者无明显症状,而仅仅是外阴皮肤黏膜颜色的变浅。如果患者因症状不明显而延误治疗,或治疗效果欠佳,病变长期发展,会导致很多的后遗症,常见的有外阴阴蒂处形成瘢痕、阴道口粘连狭窄,患者常常伴有性心理障碍。无奈的是,只有活检组织学病理诊断是确诊的唯一金标准。目前,外阴硬化性苔癣的病变原因,发病机制都还不清楚,研究发现本病的发生可能与很多因素有关,包括：自身免疫因素、遗传因素、新陈代谢、感染因素、内分泌因素及局部因素等。
目前对VLS没有确切有效的治疗方法,多年前,因为VLS可以发展成外阴癌,作为一种癌前病变,主张手术切除,但是手术后所带来的外阴的瘢痕形成等并发症更加严重的影响了病人的生活质量。目前认为,虽然VLS是一种癌前病变,但是癌变率低,只有大约3%-5%的患者可能发展为外阴鳞状细胞癌,治疗方案的选择时要充分权衡利弊,主张首选保守治疗。虽然对外阴硬化性苔癣的临床治疗也进行了广泛的探索,但是直到现在,也没有发现一种确切有效的治疗药物,国际上认为局部应用糖皮质激素涂擦患处,虽然有一定的疗效,但是需要一天涂抹多次,使用起来很不方便,并且停药一段时间后还会发作,需要继续重复使用。用的时间长了还会引起外阴皮肤粘膜萎缩而加重病变,有的还有皮肤刺激症状。鉴于西药治疗VLS的诸多弊端,我们有必要将研究目光转向毒副作用少、标本兼治、不会产生药物依赖性的传统医药。我们在积山东大学齐鲁医院对本病四十余年的临床与实验研究的基础上,自拟阴痒洗剂治疗本病,以研究其临床疗效和作用机理。
研究目的
本研究旨在观察阴痒洗剂治疗外阴硬化性苔癣的临床疗效,并进一步探讨其作用机理,以期探索出一条治疗外阴硬化牲苔癣的有效途径,为阴痒洗剂的推广应用提供有力的临床与实验依据。
研究方法
外阴硬化性苔癣的临床诊断、中医辨证分型标准及疗效评定标准均参考《中医妇科学》、《妇产科学》和《中药新药临床研究指导原则》制定。确诊为外阴硬化性苔癣的116例患者入选本研究,随机分为两组,分别是阴痒洗剂组58例,丙酸氯倍他索软膏西药组58例；另取38名正常妇女的空腹血清和13名正常妇女的外阴皮肤做正常对照组。重点观察阴痒洗剂治疗组和西药对照组治疗前后的外阴瘙痒情况、外阴皮肤粘膜的病变范围和颜色变化情况,检测治疗前后血清T细胞亚群CD3+、CD4+、CD8+和免疫球蛋白IgG、IgA、IgM的变化,观察治疗前后局部组织骨桥蛋白的表达情况,并观察治疗前后的组织病理学变化。
结果
1.阴痒洗剂能有效改善外阴硬化性苔癣患者的临床症状和体征,各中医辨证分型间疗效未见明显差异。
2.阴痒洗剂治疗组总有效率82.76%,西药对照组总有效率41.38%,两组比较差异有统计学意义(P<0.05),治疗组疗效优于西药对照组。
3.治疗前患者的血清CD3+、CD4+下降,CD8+升高,CD4+/CD8+下降,与正常对照组比较,差异有统计学意义(P<0.05)。阴痒洗剂治疗组治疗后CD3+、CD4+升高,CD8+下降,CD4+/CD8+升高,与正常对照组比较无显著性差异(P>0.05)。西药对照组于治疗前后各项指标比较无显著性差异(P>0.05)。两组治疗后各项指标比较差异有统计学意义(P<0.05)。
4.治疗前患者血清IgG水平下降,与正常对照组比较,差异有统计学意义(P<0.05)。阴痒洗剂治疗组治疗后IgG升高,与正常对照组比较无显著性差异(P>0.05)。西药对照组于治疗前后IgG水平比较无显著性差异(P>0.05)。两组治疗后IgG水平比较差异有统计学意义(P<0.05)。
5.治疗前患者病变皮肤的OPN表达显著增高,与正常对照组比较差异有统计学意义(P<0.05)。阴痒洗剂治疗组治疗后OPN表达降低,与正常对照组比较无显著性差异(P>0.05)。西药对照组于治疗前后各项指标比较均无显著性差异(P>0.05)。
6.组织病理学发现,治疗组治疗后表皮角化过度减轻,棘细胞层数增多,上皮脚出现,基底层空泡变性消失或明显改善,黑素细胞增多,真皮乳头恢复,真皮水肿和胶原均质化带消失或明显改善,炎细胞浸润明显减少。
结论
1.阴痒洗剂可以缓解外阴硬化性苔癣的症状、体征,临床疗效满意。
2.免疫功能的改变可能与VLS的病变相关,阴痒洗剂的治疗作用可能与免疫调节有关。
3.0PN可能与阴痒洗剂的免疫调节治疗作用有关。
意义
本研究为治疗外阴硬化性苔癣提供了一种有效的中药-阴痒洗剂,并初步探讨了阴痒洗剂的可能作用机理和骨桥蛋白在阴痒洗剂治疗中的作用,从而为阴痒洗剂的进一步推广应用提供有力的临床与实验依据。
Part I Study on the relationship of osteopontin and matrix metalloproteinases-9in vulvar diseases
Background and significance
Vulvar diseases mainly include vulvar carcinoma, vulvar intraepithelial neoplasia (VIN) and vulvar non-neoplastic epithelial disorders (VNED). VNED includes vulvar lichen sclerosus (VLS), vulvar squamous cell hyperplasia and other dermatoses. Vulvar squamous cell carcinoma (VSCC), an invasive carcinoma, is constructed by squamous cells of different degrees of differentiation. Squamous cell carcinomas (SCCs), the most common vulvar carcinomas, account for about90%of all vulvar malignancies and3%-5%of all gynaecological carcinomas. However, over the age of75the incidence of vulvar carcinoma peaks to approximately25%, making it as common as cervical carcinoma. VSCC represents an important medical challenge worldwide whose incidence is increasing at an alarming rate in young females. The diagnosis of VSCC is often delayed and the clinical management is very difficult. Surgery is considered to be the cornerstone of treatment of VSCC, and radio-and/or chemotheraphy only play a limited role. However, postoperative complications of vulvar impact seriously on the patient's quality of life. Therefore, the research work on the progression and prognosis of VSCC is very important.
Vulvar intraepithelial neoplasia (VIN) and vulvar lichen sclerosus (VLS) are all regarded as precancerous lesions. VIN, less seen in the vulva, is a histological diagnosis based on loss of squamous epithelial maturation associated with enlarged, hyperchromatic, pleomorphic nuclei and increased, usually atypical mitoses. There are two types of VIN according to the newest separate criteria:the usual type and the differentiated type. These two types are all regarded as pre-malignant lesions. The former is related to high-risk human papillomavirus and primarily affects younger patients. The latter is not related to HPV. It occurs in older women and leads to mostly differentiated keratinizing squamous cell carcinoma. This type relates to chronic inflammatory vulvar conditions such as VLS and accounts for the majority of all VSCC. VLS, often seen in the department of gynecology, is a chronic inflammatory skin disease, runing a relapsing and remitting course, and the management is difficult. The common symptom is pruritus, micturition difficult, algopareunia and burning sensation. At least one third of patients may be asymptomatic. Typically, the lesions are white plaques and papules, often with areas of erythema, ecchymosis, hyperkeratosis, pallor, fissuring, telangiectasia, hyperpigmentation, bullae, excoriation, oedema and/or ulceration. The lingering effects, most often encountered in long-term cases of VLS, include scarring, narrowing of the introitus which can make intercourse impossible, as well as widespread psychosexual disorder. The risk of developing squamous cell carcinoma of the vulva approaches3%-5%in women with VLS, which therefore is regarded as a pre-malignant lesion.
Early detection and treatment of VIN and VLS may prevent development of VSCC and improve its prognosis. The oncogenesis of VLS to VSCC and its connection with VIN also remains to be elucidated. In order to prevent the progression of VSCC, it is important to find a way to predict the development of vulvar precancerous lesions.
Progress of malignant tumors rely on the regulation of the extracellular matrix protein, which helps regulate the transfer of genetic modification and physiological changes, and OPN is such an extracellular matrix protein. Osteopontin is a highly phosphorylated glycoprotein and has been shown to be synthesized in a variety of tissues and cells and secreted into body fluids. OPN stimulates various signal transduction pathways through binding to its receptors, such as integrins and CD44variants, and therefore plays an active role in many physiological and pathological processes. It has also been shown that increased OPN expression correlates with tumor progression and metastasis in breast cancer, lung cancer, colon cancer, and in others cancers. It has been shown that OPN enhances the abilities of mobility and chemical invasiveness of malignant tumor cells possibly through regulating the activities of MMP-9, which degradate extracellular matrix. MMP-9is the largest and most important proteases of the matrix metalloproteinase family and plays an important role in the process of extracellular matrix degradation. MMP-9has been shown to be secreted in a variety of cells, such as hematopoietic stem cells, neutrophils, endothelial cells and a variety of tumor cells. The main role of MMP-9is to degrade the basement membrane and extracellular matrix, maintaining physiological homeostasis of extracellular matrix process. It has been shown that MMP-9degradation of extracellular matrix, promoting tumor angiogenesis, thereby affecting the outcome and prognosis of cancer patients in the process of tumor invasion and metastasis.
However, to the best of our knowledge, the expression of OPN and MMP-9in VSCC, VIN and VLS has not yet been elucidated so far.
In this study, we detect the expression and clinical significance of OPN and MMP-9in VSCC, VIN and VLS by the immunohistochemical method and chromogenic in situ hybridization.
Objective
We aimed to observe the expression of OPN and MMP-9in VSCC, VIN, VLS, and control. We investigated the role of OPN and MMP-9in the development of vulvar diseases and canceration, and investigated the relationship of OPN and MMP-9to the clinicopathologic factors and prognosis. The clinical-pathological prognostic factors of VSCC were investigated.
Methods
For immunohistochemical evaluation and chromogenic in situ hybridization, archival formalin-fixed, paraffin-embedded vulvar specimens from2002to2007were retrieved from the Department of Pathology and the Department of Dermatology at the Qilu Hospital of Shandong University. Samples from25VSCC,21VIN,21VLS and13control were collected to detect the expression of OPN and MMP-9. Immunohistochemical staining for OPN and MMP-9were performed using the streptavidin-peroxidase method.We analyzed the clinical pathology data of the patients with VSCC. All statistical analyses were performed with SPSS13.0statistical software. The x2test was performed to examine the association between OPN, MMP-9and variable clinicopathologic factors. Kaplan-Meier method was used to calculate the survival curves, and log-rank test was used to compare the statistical significance of difference between the survivals of patient subgroups.
Results
1. OPN and MMP-9expression increase with the degree of malignancy of vulvar lesions by the immunohistochemical method and chromogenic in situ hybridization (P <0.05).
2. OPN staining was found in the extracellular matrix in VSCC and VIN by the immunohistochemical method. The expression of MMP-9was notable increase in normal epidermis adjacent to VSCC when compared with normal skin by the immunohistochemical method.
3. OPN overexpression and high MMP-9were no significantly associated with tumor relapse by the immunohistochemical method and chromogenic in situ hybridization (P<0.05).
4. Univariate survival analysis showed that patients with OPN overexpression had a significantly poor overall survival at5years after operation by the immunohistochemical method (P=0.033).
5. Univariate survival analysis showed that patients with MMP-9overexpression had a significantly poor overall survival at5years after operation by chromogenic in situ hybridization (P=0.037).
6. Univariate survival analysis showed that patients with OPN overexpression had a significantly poor disease-specific survival at5years after operation by the immunohistochemical method (P=0.048)
7. The x2test showed that OPN overexpression was significantly associated with high MMP-9by the immunohistochemical method (P=0.000).
8. The x2test showed that OPN overexpression was significantly associated with high MMP-9by chromogenic in situ hybridization (P=0.001).
Conclusion
1. OPN and MMP-9are involved in the development of the vulvar diseases and the malignant progression of VSCC.
2. OPN protein overexpression is significantly associated with poor survival and may have clinical potential as a promising predictor to identify individuals with poor prognostic potential.
3. MMP-9may represent an early stage of malignant transformation in VSCC, while OPN perhaps a late stage.
Part Ⅱ Study of Yinyang lotion in the treatment of vulvar lichen sclerosus
Background
Vulvar lichen sclerosus (VLS), often seen in the department of gynecology, is a chronic inflammatory skin disease, runs a relapsing and remitting course, and the management is difficult. VLS occurs at all ages, and has a bimodal peak incidence in menopausal women. The common symptom is pruritus, micturition difficult, burning sensation and algopareunia. At least one third of patients may be asymptomatic. Typically, the lesions are white plaques and papules, often with areas of erythema, ecchymosis, hyperkeratosis, pallor, fissuring, telangiectasia, hyperpigmentation, excoriation, oedema and/or ulceration. The lingering effects, most often encountered in long-term cases of VLS, include scarring, narrowing of the introitus which can make intercourse impossible, as well as widespread psychosexual disorder. The risk of developing squamous cell carcinoma of the vulva approaches3%-5%in women with VLS. The histological diagnosis is the only method to make a definite diagnosis. Up to now, the pathogenesis of VLS is incompletely understood. The autoimmunity, endocrine factor, enetic factor, metabolism, infection and local factor may be involved in it. The early diagnosis and treatment for VLS is a challenge for gynaecologists. In the past few years, scholars investigated the aetiology, pathogenesy and management of VLS, however, extensively approved management. The local corticosteroids is regarded as the first choice, but its side effects, including atrophia and skin stimulus, make the treatment to run into a predicament. The conbined treatment of traditional Chinese medicine and western medicine was used by internal scholars and got satisfactory results. We used Yinyang lotion to treat VLS on the basis of clinical and empirical study for more than40years in Qilu Hospital of Shandong University, and investigated its clinical curative effect and mechanism of action.
Objective
To observe the clinical curative effect of Yinyang lotion on VLS, and investigate the mechanism of action, so as to explore an effective way for the prevention and treatment of VLS, and provide strong theoretical and practical basis for the wide administration of Kebai cream.
Methods
We referred to the medicine teaching material edited by the government (Chinese Gynaecology, Obstetrics and Gynecology), Practical Obstetrics and Gynecology (second edition) and The Clinical Guidlines for the New Chinese Medicine,(Enacted by the Department of Health of China in1993and2002), and instituted the clinical diagnosis, the criterion of differentiation of symptoms and signs for classification of syndrome with traditional Chinese medicine and evaluation criterion of curative effect of VLS.
116patients with VLS were divided into case group and control group randomly. The case group (58patients) was treated with Yinyang lotion while the control group (58patients) was treated with clobetasol propionate ointment. The serum from38healthy women and vulvar tissue from13normal women were collected as normal control. The changes of the symptoms and signs such as pruritus, depigment and atrophy, were followed up during the period of the management. We detected the serum of the patients and the healthy women for T cell subsets (mainly CD3+, CD4+, CD8+T cells), IgG, IgA, IgM. The expression of osteopontin (OPN) and the histomorphology change in the prior and post-treatment were observed.
Results
1. The symptoms and signs of the case group, such as itching, depigment and atrophy, were greatly improved after treatment. And the validity among the types of case group was found no significant difference.
2. The total effective rate of case group is82.76%, while the control group is41.38%. By comparation, a statistical meaning can be educed (P<0.05), which is that the curative effect of the case group is better than the control group.
3. the expression of CD3+, CD4+and CD4+/CD8+T cells decrease significantly, while the significant increases of CD8+T cells were observed before the treatment (P <0.05). However, significant increases of CD3+, CD4+and CD4+/CD8+T cells, and the significant decreases of CD8+T cells were observed after the treatment with Yinyang lotion (P<0.05). There was no difference in results for T cells expression in the prior and post-treatment with clobetasol propionate ointment (P>0.05). After treatment, the significant difference in the expression of T cells between case group and control group was observed (P<0.05).
4. Significant decreases in the level of IgG were observed before treatment(P< 0.05). However, significant increases of IgG were detected after the treatment with Yinyang lotion (P<0.05). There was no difference in results for IgG in the prior and post-treatment with clobetasol propionate ointment (P>0.05). After treatment, the significant difference in level of IgG between case group and control group was observed (P<0.05).
5. By immunohistochemistry, significant decreases of OPN immunostaining cells were observed after the treatment with Yinyang lotion (P<0.05). There was no difference in results for OPN expression in the prior and post-treatment with clobetasol propionate ointment (P>0.05).
6. Observation by light microscope, after the treatment with Yinyang lotion, showed that lessened hyperkeratinization in the epidermis, more prickle cell layer, appearing rete ridges, improved obviously basal keratinocyte hydropic degeneration and vacuolization, melanocytosis, restored dermal papilla, obvious improvement or loss of edema and homogenization of collagen in the upper dermis, lessened obviously lymphocytic infiltration.
Conclusion
1. Yinyang lotion has a good clinical curative effect, and is an ideal medicine for theraphy of VLS.
2. Changes in immune function may be associated with VLS lesions, Yinyang lotion therapeutic effect may be related to immune regulation.
3. The mechanism of Yinyang lotion may adjust the immune function, OPN may be involved in this process.
引文
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