Role of normal cervical stem cells in HPV induced initiation of cervical cancer

虽然HPV已被确定为几乎所有宫颈癌的病原体，但大多数宫颈HPV感染不会导致宫颈癌。只有1%的病例进展为高度异型增生和癌症。大多数宫颈HPV感染是短暂的非肿瘤性生产性病毒感染，在一年内消失。普遍认为，高危型HPV感染的持续存在是宫颈癌发生发展的必然状态。决定感染持续性的关键因素及其与免疫状态正常的妇女患宫颈癌风险增加的相关性在很大程度上仍然未知。免疫力受损的妇女，如艾滋病毒/艾滋病患者，是否存在这些因素也不得而知，但在艾滋病毒感染者中，宫颈癌的发病率有所上升。持续性感染可能只发生在特定宫颈细胞的靶向感染后，可能具有干细胞特性。为了验证这一想法，我们利用从合作人类组织网络获得的新鲜人类组织宫颈上皮组织。我们的实验室成功地开发了一个协议，以分离宫颈上皮细胞，并确定一个表型不同的亚群与干细胞预期的功能特性。初步数据表明，这种细胞部分，起源于宫颈上皮基底层具有10倍的能力，结合乳头瘤病毒样颗粒（VLP），因此意味着这些细胞可能会表达更多的乳头瘤病毒结合位点，促进病毒感染，如果病毒的目标。为了优化重现干细胞原位功能条件的环境，我们还开发了一种宫颈外器官培养模型，使我们能够建立和监测HPV假病毒在新鲜宫颈组织中的结合和感染。培养的宫颈组织经历快速的细胞解离和更分化的顶端上皮层的脱落。相反，基底细胞仍然附着在基底膜上，假定上皮干细胞居住在基底膜上。我们已经观察到，随着时间的推移，基底层内只有非常有限数量的细胞保持被假病毒感染，假病毒使用红色荧光蛋白作为报告基因。我们计划使用FACs分析来表征感染的细胞，以确定它们是否确实是宫颈干细胞。我们计划使用在NCI的John Schiller实验室开发的弗林蛋白酶裂解假病毒来提高感染效率。先前观察到，在这种全组织贴装条件下，以前静止的基底细胞可以变得增殖并再生整个上皮，从而保持通常在体内观察到的组织结构和细胞异质性。据推测，当在暴露基底膜的上皮层的机械创伤后体内发生HPV感染时，所产生的顶端层的再生最终可能引发瘤形成。导致这种发展的条件目前正在优化中，一旦实施，将进一步模拟体内HPV感染，并帮助我们了解宫颈癌的发生。我们计划通过使用全球遗传方法寻找新的标志物来进一步表征宫颈干细胞群体，该方法将改善干细胞分离，并研究这些细胞在生产性病毒感染进展到病毒诱导的癌前病变和宫颈癌中的作用。本研究旨在提高我们对HPV诱导的宫颈癌发生的理解，并有助于开发针对宫颈癌的新策略。

Though HPV has been established as the etiologic agent for virtually all cervical cancers, the majority of cervical HPV infections do not lead to cervical cancer. Only 1% of cases progress to high grade dysplasia and cancer. The majority of cervical HPV-infections are transient non-neoplastic productive viral infections which disappear within a year. It is generally accepted that persistence of high risk HPV infection is an imperative state in the development of cervical neoplasia. The crucial factors determining persistence of infection and its correlation with an increased risk for cervical cancer in women with normal immune status are still largely unknown. These factors in women with compromised immunity such as in HIV/AIDS is also unknown, but there is an increased prevalence of cervical cancer in the HIV population. Persistent infection may only occur upon targeted infection of specific cervical cells, possibly with stem cell properties. In order to test this idea, we utilize fresh human tissue cervical epithelial tissue obtained from the Cooperative Human Tissue Network. Our laboratory successfully developed a protocol to isolate cervical epithelial cells and to identify a phenotypically distinct subpopulation with functional properties expected for stem cells. The preliminary data show that this cell fraction, originating from the basal layer of the ectocervical epithelium possesses an 10 fold increased capacity to bind papillomavirus-like particles (VLP), thus implying that these cells may express an increased number of papillomavirus binding sites facilitating viral infection if targeted by the virus. In an effort to optimize an environment that recapitulates the conditions under which stem cells function in situ, we have also developed an ectocervical organ culture model that allows us to establish and monitor HPV pseudovirus binding and infection in fresh cervical tissue. Cervical tissue maintained in culture undergoes rapid cell dissociation and exfoliation of the more differentiated, apical epithelial layers. In contrast, the basal cells remain attached to the basement membrane, where presumed epithelial stem cells reside. We have observed that only a very limited number of cells within the basal layer remains infected over time by pseudovirus using red fluorescent protein as a reporter gene. It is our plan to characterize the infected cells using FACs analysis to determine whether they are indeed cervical stem cells. We plan to use a furin cleaved pseudovirus developed in John Schiller's lab at NCI to increase the efficiency of infection. It was previously observed that the formerly quiescent basal cells could become proliferative and regenerate the entire epithelium under such whole tissue mount conditions, preserving the tissue architecture and cellular heterogeneity normally observed in vivo. It is postulated that when HPV infection occurred in vivo following a mechanical trauma to epithelial layer exposing the basal membrane, the resulting regeneration of apical layers could ultimately spark neoplasia. The conditions that lead to such development are currently under optimization and upon implementing, will further mimic in vivo HPV infection and assist our understanding of cervical carcinogenesis. We plan to further characterize the cervical stem cell population by searching for new markers using a global genetic approach which will improve stem cell isolation and to investigate the role of these cells in progression of a productive viral infection to virus induced pre-malignant lesions and cervical cancer. This study aims to improve our understanding of HPV induced cervical carcinogenesis and contribute to the development of novel strategies designed to target cervical cancer.