基于上皮器官筏模型探讨复方狼毒方对尖锐湿疣发病环节HPVE6E7下调抑瘤因子p53、Rb的干预作用

Condyloma acuminatum is the most common disease caused by HPV infection, which is refractory and easy to relapse.While HPV infection is the primary factor leading to cervical cancer.HPVE6E7 gene cause papilloma hyperplasia in host epithelial tissue by down-regulating the expression of tumor suppression factor p53 and Rb protein, which appear even malignant transformation.Early research has shown that compound Euphorbia and its monarch drug E. Ebracteolata Hayata can treat effectively multiple verruca caused by HPV infection in external use, inhibit Hela cell proliferation and induce apoptosis.Due to the fact that HPV life cycle highly depend on epithelial tissue differentiation, this study proposed that first construct plasmid containing HPV18 full-length gene which is transfected to primary human keratinocytes, then build a Organotypic raft model in which HPV life cycle can be expressed completely , the mechanism of compound Euphorbia in treating condyloma externally is explored, from the pathogenesis in that HPVE6 E7 gene down-regulate the expression of tumor suppression factor p53 and Rb protein, and in this process the Southern blotting, western blotting, PCR, in situ hybridization and immunohistochemical technique are used.The method improve and verify the TCM theory of “combat poison with poison”, enrich the external therapy of traditional Chinese medicine.In this topic function characteristics and molecular mechanism of treatment with compound Euphorbia is explored and a new method of TCM to treat the diseases caused by HPV infection has been shown.

尖锐湿疣(CA)是HPV感染所致疾病中最常见的疾病，难治易于复发。同时HPV感染也是导致宫颈癌的首要因素。HPVE6E7基因通过下调抑瘤因子p53、Rb蛋白表达导致宿主上皮组织乳头瘤样增生，甚至出现恶性转化。前期研究表明复方狼毒方及君药月腺大戟外用可有效治疗HPV感染所致多发性疣赘，抑制Hela细胞增殖和引起凋亡。由于HPV生活周期高度依赖上皮组织分化，本研究拟通过含有HPV18全长基因的质粒转染原代角质形成细胞，构建完整表达HPV生活周期的上皮器官筏模型，采用DNA印迹杂交、免疫印迹、PCR、原位杂交、免疫组织化学等技术，从HPVE6E7下调抑瘤因子p53、Rb的发病环节，探讨复方狼毒方外用治疗CA的作用机制。复方狼毒方治疗CA是对中医“以毒攻毒”理论的挖掘、提高和验证,丰富了中医外治疗法。本课题探求复方狼毒方治疗CA的作用特点和分子机制，提出了中医药治疗人乳头瘤感染症的新途径、新方法。

尖锐湿疣(CA)是HPV感染所致疾病中最常见的疾病，难治易于复发。同时HPV感染也是导致宫颈癌的首要因素。HPVE6E7基因通过下调抑瘤因子p53、Rb蛋白表达导致宿主上皮组织乳头瘤样增生，甚至出现恶性转化。前期研究表明复方狼毒方及君药月腺大戟外用可有效治疗HPV感染所致多发性疣赘，抑制Hela细胞增殖和引起凋亡。本研究从HPVE6E7基因通过下调抑瘤因子p53、Rb蛋白表达的发病环节探讨复方狼毒方外用治疗CA的作用机制。由于HPV生活周期高度依赖上皮组织分化，本研究首先构建HPV18稳定转染的角质细胞系，用HPV18转染的单层角质细胞模拟HPV潜伏期感染，然后成功建立了HPV18转染的上皮器官筏模型，用HPV18转染的上皮器官筏模型模拟HPV生活周期晚期相。对HPV潜伏期感染及生活周期晚期相感染分别用单方狼毒（月腺大戟）水提取物、醇提取物，复方狼毒方水提取物、醇提取物4种成分干预，运用qPCR、免疫荧光、免疫印迹法等分子生物学技术检测细胞增殖、HPV DNA复制，P53、pRb mRNA及蛋白表达、HPV L1表达，结果表明对HPV潜伏期感染及HPV生活周期晚期相感染,复方狼毒方及君药月腺大戟的水提取物、醇提取物均可抑制角质细胞增殖，同时明显抑制HPV DNA复制（抑制E6基因复制及下调L1基因表达），上调P53、pRb表达（P53、pRb mRNA及蛋白表达均上调）。四种提取物比较以复方狼毒方醇提取物效果最为明显。本研究从基因分子水平验证了复方狼毒方及其君药月腺大戟治疗尖锐湿疣的作用机制为通过影响E6、E7基因表达上调肿瘤抑制因子p53 、pRb蛋白， 从而抑制宿主细胞的增殖及HPV病毒核酸复制，本研究为中医药治疗人乳头瘤感染症提供了新途径、新方法，也是对中医“以毒攻毒”理论的挖掘、提高和验证。