【作者】 李育竹；

【导师】 李淑玲；

【作者基本信息】 山东大学 ， 中西医结合临床， 2009， 博士

【摘要】 研究背景外阴皮肤和粘膜的病变主要包括外阴上皮内非瘤样变、外阴鳞状上皮内瘤样变和外阴癌。外阴上皮内非瘤样变是女性外阴皮肤和粘膜组织色素改变和变性的一组慢性疾病,包括外阴硬化性苔癣、外阴鳞状上皮细胞增生和其他皮肤病。外阴硬化性苔癣是妇科常见的慢性炎症性皮肤病,是一种较难治疗的、易复发的疾病。最常见的症状是顽固性外阴瘙痒,有些患者表现为排尿困难、性交痛和外阴烧灼感,另一些患者可以无症状。病变特点为外阴萎缩、皮肤颜色变白、发亮、皱缩、变脆、弹性差,常伴有皲裂和破溃难愈。长期患病会引起一系列后遗症,包括外阴形成瘢痕、阴道口狭窄以及普遍存在的性心理障碍,大约4%-6%的患者可能发展为外阴鳞状细胞癌,因此有人将其视为外阴癌前病变。外阴鳞状上皮内瘤样变是一种发生于女性外阴的较少见的疾病,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。组织学表现以基底膜以上表皮细胞成熟紊乱、胞核异常为特点。根据最新制订的分类标准,按照有无人乳头瘤病毒(humanpapillomavirus,HPV)感染将其分为寻常型和分化型二种,前者与高危型HPV病毒感染有关;而后者与HPV感染无关,并可能是外阴硬化性苔癣向外阴鳞状细胞癌进展的一个很短暂的上皮病变过程。这二种外阴鳞状上皮内瘤样变均被认为是癌前病变。而外阴鳞状细胞癌是由不同分化程度的鳞状上皮细胞构成的浸润性癌,是最常见的外阴癌,占外阴恶性肿瘤的80%-90%。其发病率虽然仅占女性生殖系统恶性肿瘤的5%左右,但在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。近年来,外阴鳞状细胞癌发病率升高,患者有渐趋年轻化倾向。目前认为其发病主要有二条途径,一与外阴长期慢性炎症刺激有关,与HPV感染无关,其癌前病变为外阴硬化性苔癣和/或分化型外阴鳞状上皮内瘤样变,主要见于老年妇女;二与高危型HPV感染有关,即寻常型外阴鳞状上皮内瘤样变为其癌前病变,其主要发病人群为年轻妇女。外阴皮肤和粘膜的病变广泛发生于各年龄阶段的妇女,而长期以来对此领域的研究相对较少,至今各种病变的发病机制均不明,导致临床治疗上存在很大困难。缺氧是某些生理、病理现象发生的根本原因,也是实体肿瘤微环境的基本特征之一,在多种恶性肿瘤及癌前病变中均可见到相关研究。缺氧导致一系列基因和蛋白的表达,可以刺激肿瘤细胞血管生成和糖酵解,上调一些生长因子以及肿瘤侵袭相关蛋白的表达,使肿瘤细胞发生恶性转化、浸润,甚至转移。缺氧诱导因子-1及其下游靶基因的表达是多种恶性肿瘤的预后因子,也预示着放化疗抵抗,并具有靶向治疗作用。然而,缺氧微环境与外阴鳞状细胞癌的发生、进展的相关性研究在国内外尚未见报道。研究目的研究缺氧相关因子和细胞增殖标记物在正常对照外阴皮肤、外阴硬化性苔癣、外阴鳞状上皮内瘤样变和外阴鳞状细胞癌中的表达及其与外阴鳞状细胞癌的临床病理资料间的相关性,探讨缺氧在外阴病变的发生和发展过程中的作用,探讨缺氧与细胞增殖的相关性,并为进一步研究可靠的外阴鳞状细胞癌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1月-2007年12月存档的石蜡组织标本,包括外阴硬化性苔癣41例、寻常型外阴鳞状上皮内瘤样变10例、外阴鳞状细胞癌25例,另取正常外阴皮肤12例,组织切片进行免疫组织化学染色检测缺氧相关因子,包括缺氧诱导因子-1α(hypoxia-induciblefactor-1α,HIF-1α)、葡萄糖转运蛋白-1(glucose transporter-1,GLUT-1)、碳酸酐酶-9(carbonic anhydrase IX,CA IX)、血管内皮生长因子(vascular endothelial growthfactor,VEGF)及细胞增殖标记物Ki-67蛋白的表达。对外阴鳞状细胞癌患者的临床病理资料进行分析,随访。结果1.GLUT-1在正常外阴皮肤中表达水平很低,明显低于外阴硬化性苔癣、外阴鳞状上皮内瘤样变和外阴鳞状细胞癌(P＜0.05),在外阴鳞状细胞癌中表达最高(P＜0.05)。2.CA IX在正常外阴皮肤和硬化性苔癣中均无表达,在外阴鳞状上皮内瘤样变和外阴鳞状细胞癌中表达增多(P＜0.05),后二者比较差异无统计学意义(P＞0.05)。3.VEGF在外阴鳞状细胞癌中表达最高,其次为外阴鳞状上皮内瘤样变(P＜0.05),明显高于硬化性苔癣和正常外阴皮肤,后二者的表达无显著性差异(P＞0.05)。4.Ki-67在外阴硬化性苔癣、外阴上皮内瘤样变及外阴鳞状细胞癌中均较正常外阴皮肤表达显著增多(P＜0.05),而后二者比较无显著性差异(P＞0.05)。5.HIF-1α在各组中均未见明确的细胞核表达。6.在各组外阴病变中,缺氧相关因子与细胞增殖标记物Ki-67之间均未见相关性;GLUT-1与VEGF的表达间均存在显著的正相关关系,而CA IX与VEGF的表达无明显相关性。7.外阴鳞状细胞癌的1年、3年及5年生存率分别为75%,70%,64%,未见明显的预后因子。结论1.缺氧参与外阴病变的发生及恶性进展,GLUT-1异常高表达可能是病变进展的早期事件,CA IX和VEGF的表达是晚期事件。2.在回顾性研究中,GLUT-1可能是比HIF-1α更敏感的内源性缺氧标记物;HIF-1α表达不稳定,可能出现假阴性表达;CA IX在外阴组织中表达不敏感。3.缺氧与细胞增殖之间未见相关性。4.外阴鳞状细胞癌是一种预后较好的恶性肿瘤,缺氧相关因子不是判断其预后的理想因子。意义缺氧与外阴病变的相关性研究在国内外尚未见报道,本研究初步探讨了缺氧相关因子在外阴鳞状细胞癌的发生、恶性进展中的作用,为此领域的进一步研究提供有益的研究基础。研究背景外阴硬化性苔癣是妇科常见的慢性炎症性皮肤病,是一种较难治疗的、易复发的疾病。本病可见于任何年龄,国外报道在青春期前和绝经后妇女中发病率较高,其发病率呈双峰态势;但国内报道以40岁左右妇女多见,其次为幼女。本病最常见的症状是顽固性外阴瘙痒,有些患者表现为排尿困难、性交痛和外阴烧灼感,另一些患者可以无症状。病变特点为外阴萎缩、皮肤颜色变白、发亮、皱缩、变脆、弹性差,常伴有皲裂和局部皮肤破损。长期患病会引起一系列后遗症,包括外阴瘢痕形成、阴道口狭窄以及普遍存在的性心理障碍,大约4%-6%的患者可能发展为外阴鳞状细胞癌。病理诊断是目前唯一的确诊方法。至今外阴硬化性苔癣的病因及发病机制尚不清楚,目前的研究认为可能与自身免疫因素、遗传因素、内分泌因素、新陈代谢、感染及局部因素等有关。外阴硬化性苔癣的早期诊断和治疗对于妇科医生、皮肤科医生、病理科医生和儿科医生都是一种挑战,需要多学科间的协作以深入研究。近年来国内外学者对外阴硬化性苔癣的病因及发病机制进行了大量深入的研究,对其临床治疗也进行了广泛的探索,但目前仍缺乏统一的治疗方法和疗效评价标准,国际上认为局部应用糖皮质激素是治疗硬化性苔癣的首选,但其引起的局部萎缩、皮肤刺激等副作用使治疗陷入困境。而国内学者应用中西医结合治疗方法进行探索,取得了可喜的研究成果。我们在积山东大学齐鲁医院对本病三十余年的临床与实验研究的基础上,自拟克白霜治疗本病,研究其临床疗效和作用机理。研究目的本研究旨在系统观察克白霜治疗外阴硬化性苔癣的临床疗效,探讨其作用机理,以期探索出一条治疗外阴硬化性苔癣的有效途径,为克白霜的推广应用提供有力的临床与实验依据。研究方法参考《中医妇科学》、《妇产科学》、《实用妇产科学》和《中药新药临床研究指导原则》制定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。将确诊为外阴硬化性苔癣的102例患者,随机分为两组,克白霜治疗组51例,丙酸氯倍他索软膏西药对照组51例;另取30名正常妇女的空腹血清和12名正常妇女的外阴皮肤做正常对照组。重点观察克白霜治疗组和西药对照组治疗前后的外阴瘙痒疼痛情况、外阴皮肤粘膜的病变范围和色素改变情况,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及超氧化物歧化酶(SOD)、丙二醛(MDA)的变化,观察治疗前后局部组织CD3、CD4、CD8、CD68和缺氧相关因子GLUT-1和VEGF的表达情况,并分别以光镜和透射电镜观察治疗前后的组织细胞形态学变化。同时了解克白霜在各中医辨证分型中的最佳适用证型,随访观察其长期用药的毒副作用和预防癌变的作用。动物实验观察克白霜对大鼠棉球肉芽肿的抑制作用和对4-氨基吡啶引起的过敏致痒的止痒作用。结果1.克白霜治疗组总有效率60.78%,丙酸氯倍他索软膏西药对照组总有效率33.33%,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。2.克白霜能有效改善外阴硬化性苔癣患者的临床症状和体征,各中医辨证分型问疗效未见明显差异。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)。克白霜治疗组治疗后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)。克白霜治疗组治疗后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.治疗前患者血清SOD水平下降、MDA水平升高,与正常对照组比较,差异有统计学意义(P＜0.05)。克白霜治疗组治疗后血清SOD水平明显升高,MDA水平降低,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SOD和MDA水平比较无显著性差异(P＞0.05)。两组治疗后SOD和MDA水平比较差异有统计学意义(P＜0.05)。6.免疫组化研究发现在治疗前患者的病变皮肤中,明显增多的CD3+、CD4+淋巴细胞以几乎相同的比例存在于真皮炎细胞浸润带中,少量CD3+、CD4+淋巴细胞存在于表皮真皮连结附近,偶见于表皮下层。CD8+淋巴细胞和单核巨噬细胞标记物CD68+细胞很少存在于病变皮肤中。两组治疗前后CD3、CD4的表达比较,差异均有统计学意义(P＜0.05);两组治疗后CD3、CD4的表达比较,差异均无统计学意义(P＞0.05)。7.治疗前患者病变皮肤的GLUT-1表达显著增多,而血管内皮细胞和周细胞的VEGF表达显著减少,与正常对照组比较差异有统计学意义(P＜0.05)。克白霜治疗组治疗后GLUT-1表达减少,VEGF表达增多,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)。8.光镜观察发现,两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。9.透射电镜观察细胞超微结构发现,外阴硬化性苔癣治疗前上皮基底膜厚薄不一,局部增厚、菲薄或断裂,半桥粒结构稀疏,可见上皮中的部分物质从断裂处突入真皮;基底细胞线粒体明显肿胀,可见嵴缺失和空泡化,有些线粒体内有膜性小体形成,黑色素颗粒减少。真皮毛细血管减少,局部管壁菲薄、迂回曲折,可见大量朝向管腔的胞质突起,多数毛细血管的管腔狭窄呈裂隙状,内皮细胞胞质中内质网肿胀,线粒体空化、嵴缺失,连接复合体无明显异常;毛细血管周细胞和真皮中大部分细胞均有明显的线粒体肿胀。克白霜治疗后,上皮基底膜厚度均匀,半桥粒丰富;基底细胞线粒体结构正常,可见趋于正常的嵴的排列,内质网无肿胀,黑素颗粒丰富。真皮毛细血管的内皮细胞胞质中线粒体结构正常,可见明显趋于正常的嵴的排列,内质网无肿胀,高尔基复合体结构正常,毛细血管的管腔未见狭窄。10.随访期间治疗组未见任何明显毒副作用,对照组部分患者可见皮肤萎缩加重、皮肤刺激。两组均未见一例恶变。11.动物实验表明,克白霜大、中剂量组与对照组比较,对大鼠棉球肉芽肿均有显著抑制作用,而西药丙酸氯倍他索软膏组无明显抑制作用,克白霜大、中剂量组与西药组比较差异有统计学意义(P＜0.05);克白霜大、中剂量组对4-氨基吡啶引起的过敏致痒有较好的止痒作用,且量效关系明显,克白霜大剂量组与西药组比较荠异无统计学意义(P＞0.05)。结论1.外阴硬化性苔癣是一种T淋巴细胞介导的炎症性的、氧化与抗氧化平衡失调的疾病,可能缺氧缺血机制参与发病。2.克白霜是治疗外阴硬化性苔癣的理想药物,临床疗效满意。3.克白霜的作用机制为调节机体免疫功能及氧化与抗氧化平衡,改善局部缺氧缺血微环境,改善局部组织细胞形态。4.随访表明克白霜长期外用无任何明显毒副作用,可预防癌变。5.克白霜药效学研究表明,克白霜有较好的抗炎、止痒作用。意义本研究为外阴硬化性苔癣提供了新的病因病机学说—缺氧缺血参与其发病,为治疗外阴硬化性苔癣提供了一种新制剂—克白霜,为制定统一的临床诊断标准、中医辨证分型标准及疗效评定标准提出了一种新方法,具有广泛的临床应用价值。更多还原

【Abstract】 Part I Study on the relationship of hypoxia and vulvar diseasesBackgroundVulvar diseases of skin and mucosa include mainly non-neoplastic epithelial disorders (NNEDs), vulvar intraepithelial neoplasia (VIN) and vulvar squamous cell carcinoma (VSCC). NNEDs, including vulvar lichen sclerosus (VLS), vulvar squamous cell hyperplasia and other dermatoses, are chronic lesions with the characteristics of depigmenting and denaturation in vulvar skin and mucosa. 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. 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 approaches 4%-6% in women with VLS, which therefore is regarded as a pre-malignant lesion. 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 (uVIN), also known as warty-basaloid, and the differentiated type (dVIN). The former is related to high-risk human papillomavirus (HPV), and the latter is not related to HPV. The dVIN perhaps is a transitory process from VLS to VSCC. The two types of VIN are all regarded as pre-malignant lesions. VSCC, an invasive carcinoma, is constructed by squamous cells of different degree of differentiation. Squamous cell carcinomas (SCCs), the most common vulvar carcinomas, account for about 80%-90% of all vulvar malignancies and 5% of all gynaecological carcinomas. However, after the age of 75 the incidence of vulvar carcinoma peaks to approximately 25%, 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. Despite a stable pattern in the incidence of vulvar carcinoma, the incidence of uVIN and vulvar carcinoma is increasing in women aged 50 years and younger. This might be due to a higher incidence of HPV infection of the genital tract and/or to an increased awareness of uVIN. VSCC develops following two different pathways, which have their own premalignant lesions. In the absence of HPV, VSCC can develop in a background of lichen sclerosus (LS), dVIN or both. The other pathway leading to VSCC is associated with HPV and the HPV-associated premalignancy is uVIN. Several factors have been linked to vulvar diseases of skin and mucosa development, but the exact pathogenesis remains to be determined, and the clinical management is very difficult.In the processes associated with both development of some physiologial or pathological phenomenon and progression to invasive disease, hypoxic microenvironment perhaps plays an important role. Hypoxia can lead to the expression of a series of genes and proteins, induce angiogenesis and glycolysis, up-regulate the expression of some growth factors and invasion-associated proteins, and cause the malignant transformation, invasion, even metastasis. The expression of hypoxia-inducible factor-1 and its target genes is the prognosis factors of many kinds of malignant tumors, and prognosticates the therapy resistance and the effect of targeted therapy. Hypoxia is associated with tumorigenesis but has not yet been identified in VSCC or in vulvar premalignant lesions.ObjectiveWe aimed to observe the expression of hypoxia induced factors such as hypoxia-inducible factor-1α(HIF-1α), glucose transporter-1 (GLUT-1), carbonic anhydrase IX (CA IX) and vascular endothelial growth factor (VEGF) and cell proliferation marker Ki-67 in control, VLS, VIN, and VSCC, and research the relationship of the expression of the proteins and clinical pathology data. We investigated the role of hypoxia in the development of vulvar diseases and canceration, and explored the correlation of hypoxia and proliferation. The clinical-pathological prognostic factors of VSCC were investigated.MethodsFor immunohistochemical evaluation, archival formalin-fixed, paraffin-embedded vulvar specimens from 2002 to 2007 were retrieved from the Department of Pathology and the Department of Dermatology at the Qilu Hospital of Shandong University. Samples from 41 LSs, 10 VINs, 25 SCCs and 12 control subjects were collected to detect the expression of hypoxia induced factors including HIF-1α, GLUT-1, CA DC and VEGF, and proliferation marker Ki-67. We analyzed the clinical pathology data of the patients with VSCC.Results1. By immunohistochemistry, GLUT-1 expression showed similar significant differences in VLS and VIN than control while immunoreactive cells in SCC were significantly more than LS and VIN (P<0.05).2. CA IX expression was negative in all control and LS sections, while similar significant differences in VIN and SCC (P>0.05).3. VSCC had significant staining of VEGF followed by VIN, LS and normal vulva. The similar immunoreaction of VEGF was showed in LS and normal vulva (P> 0.05).4. Ki-67 expression showed similar significant differences in VIN and VSCC (P> 0.05) while immunoreactive cells in control was significantly less than vulvar diseases (P<0.05).5. Nuclear staining of HIF-1αwas not found in any tissue.6. In all groups, the correlation of the expression of hypoxia induced factors and proliferation marker Ki-67 was not seen. The direct correlation of GLUT-1 and VEGF can be obviously seen, but the correlation of CA IX and VEGF was not detected.7. The survival rate of the patients with VSCC in 1, 3, 5 years is 75%, 70%, 64% respectively. No prognosis factor was detected.Conclusion1. Hypoxia is involved in the development of the vulvar diseases and the malignant progression of VSCC. GLUT-1 may represent an early stage of malignant transformation in vulvar diseases, while CA IX and VEGF perhaps a late stage.2. In the retrospective studies, GLUT-1 may be a more sensitive endogenous hypoxia marker than HIF-1α. HIF-1αis instable and a false-negative expression can be seen. CA IX is not sensitive hypoxic index in vulvar tissues.3. A correlation was found between hypoxia and proliferation.4. Vulvar squamous cell carcinoma is a invasion malignant tumor, whose prognosis is better. Hypoxia related factors arenot ideal prognostic factors of VSCC. Part II Study of Kebai cream in the treatment of vulvar lichen sclerosusBackgroundVulvar 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 prepubertal girls and menopausal women, but in the internal reports, it occurs mainly in the fourth decades, followed by prepubertal girls. 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 approaches 4%-6% 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, genetic factor, endocrine factor, metabolism, infection and local factor may be involved in it. The early diagnosis and treatment for VLS is a challenge for gynaecologists, dermatologists, pathologists and pediatricians. For the past few years, scholars investigated the aetiology, pathogenesy and management of VLS, however, extensively approved management and evaluation criterion of curative effect is absent. 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 Kebai cream to treat VLS on the basis of clinical and empirical study for more than 30 years in Qilu Hospital of Shandong University, and investigated its clinical curative effect and mechanism of action.ObjectiveTo observe the clinical curative effect of Kebai cream 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.MethodsWe 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 in 1993 and 2002), 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.102 patients with VLS were divided into case group and control group randomly. The case group (51 patients) was treated with Kebai cream while the control group (51 patients) was treated with clobetasol propionate ointment. The serum from 30 healthy women and vulvar tissue from 12 normal 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 and erythrocuprein (SOD), malonaldehyde (MDA). The expression of CD3, CD4, CD8, CD68 and hypoxia induced factors (glucose transporter-1 (GLUT-1) and vascular endothelial growth factor (VEGF)) and the histomorphology change in the prior and post-treatment were observed. Meanwhile, the best adaptive type and the toxic and side effect after long-term using of the Kebai cream were researched. Moreover, we followed up to investigate its effect to prevent the canceration. The animal experiment aimed to research the pharmacodynamics of Kebai cream.Results1. The total effective rate of case group is 60.78%, while the control group is 33.33%. By comparation, a statistical meaning can be educed (P<0.05), which showed that the curative effect of the case group is better than the control group.2. 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.3. Significant decreases in the expression of CD3+, CD4+ and CD4+/CD8+ T cells, 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 Kebai cream (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 Kebai cream (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. Significant decreases in the level of SOD, while the significant increases of MDA were observed before the treatment (P<0.05). However, significant increases of SOD, and the significant decreases of MDA were observed after the treatment with Kebai cream (P<0.05). There was no difference in results for SOD and MDA in the prior and post-treatment with clobetasol propionate ointment (P>0.05). After treatment, the significant difference in the expression of SOD and MDA between case group and control group was observed (P<0.05).6. Using immunohistochemistry, before treatment, significant numbers of CD3+ and CD4+ lymphocytes were observed in the dermal band of inflammatory cells in approximately equal proportions. Less numerous CD3+ and CD4+ lymphocytes also occurred adjacent to the dermoepidermal junction and occasionally in the lower epidermis. Less numbers of CD8+ lymphocytes and cells staining with the monocyte/macrophage marker CD68 were detected. Significant difference in the expression of CD3 and CD4 in the prior and post-treatment in both groups were observed (P<0.05). There was no difference in the expression of CD3 and CD4 after treatment between both groups (P>0.05).7. By immunohistochemistry, significant increases in the expression of GLUT-1, while the significant decreases of VEGF staining in the endothelial cells and pericytes associated with microvessels were observed before treatment (P<0.05). However, significant decreases of GLUT-1 immunostaining cells, and the significant increases of VEGF immunostaining cells were observed after the treatment with Kebai cream (P<0.05). There was no difference in results for GLUT-1 and VEGF expression in the prior and post-treatment with clobetasol propionate ointment (P>0.05).8. Observation by light microscope, after the treatment with Kebai cream, 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.9. Ultrastructural changes in VLS tissue showed that the thickness of the basement membrane (BM) of the epithelium differed as partly thickening, thinning or breaking. There were sparse hemidesmosomes on the BM; some substances in the epithelium intruded into the dermis from BM breakage. Mitochondria of basal cells were swollen, with loss of cristae and vacuolization. Morphological abnormalities in dermal blood vessels included capillary loss; a thin and convoluted vessel wall dependent on the region; cytoplasm protruding towards lumens; and narrowed or slit-shaped lumens. Mitochondria of vascular endothelial cells swelled with loss of cristae, and the rough ER (rER) revealed luminal swelling and ribosomal detachment. Junctional complexes did not display apparent abnormalities. Mitochondria of pericytes and most dermal cells were swollen. Myelin-like bodies emerged as intra-mitochondrial dense deposits in involved epidermal or dermal cells. After the treatment with Kebai cream, the tissue revealed the uniform BM of the epithelium, and abundant hemidesmosomes and melanin granules. In the cytoplasm of basal cells and vascular endothelial cells, mitochondria displayed well-defined cristae and membrane integrity; rER displayed parallel rows and attached ribosomes. Vascular lumens were well-developed.10. No toxic and side effect was seen in the case group, while adermotrophia and skin stimulus can be observed in the control group, and no canceration was seen in both groups during follow up.11. The animal experiment showed that the inhibitory action can be seen in the treatment of Kebai cream with macro- and meta-dosage and the control group, while no obvious inhibitory action in the treatment of clobetasol propionate. There was no difference in the Kebai cream with macro- and meta-dosage group and the clobetasol propionate group (P>0.05). The better action of relieving itching (itching caused by 4-aminopyridine) can be seen in the treatment of Kebai cream with macro- and meta-dosage group. And the dose-effect relationship is great. There was no difference in the Kebai cream with macro-dosage group and the clobetasol propionate group (P >0.05).Conclusion1. VLS is a inflammatory skin disease, in which the dysequilibrium between oxidation and antioxidation can be seen. Hypoxia-ischemia (HI) may be involved in the pathogenesis of VLS.2. Kebai cream has a good clinical curative effect, and is an ideal medicine for theraphy of VLS.3. The mechanism of action of Kebai cream is to adjust the immune function, promote the balance between oxadation and antioxadation, improve the local hypoxia-ischemia microenvironment and the histomorphology of vulvar tissue.4. Follow up showed that Kebai cream can prevent canceration, and no toxic and side effect was found after long periods of administration.5. The pharmacodynamics showed that Kebai cream had better effect of antiinflammatory and relieving itching.更多还原