Ex vivo 2D and 3D HSV-2 infection model using human normal vaginal epithelial cells.
Ex vivo 2D and 3D HSV-2 infection model using human normal vaginal epithelial cells.
复制标题
使用人类正常阴道上皮细胞的 ExVivo 2D 和 3D HSV-2 感染模型
DOI：
10.18632/oncotarget.14840
复制
发表时间：
2017-02-28
期刊：
影响因子：
--
通讯作者：
Li H
中科院分区：
文献类型：
--
作者：
Zhu Y;Yang Y;Guo J;Dai Y;Ye L;Qiu J;Zeng Z;Wu X;Xing Y;Long X;Wu X;Ye L;Wang S;Li H
单纯疱疹病毒2型（HSV-2）感染人类生殖器粘膜并建立终身潜伏感染。建立基于人体细胞的微生理系统是病毒生物学和抗病毒药物研究的迫切需要。几十年来，正常上皮细胞体外培养系统的缺乏是其发展的障碍之一。本研究采用共培养系统建立了人正常阴道上皮细胞（HNVEC）培养。然后在规定的培养条件下快速稳定地增殖HNVEC细胞。HNVEC细胞表现为正常的二倍体核型，在基质三维（3D）培养中形成明确的极化球体，而恶性细胞（HeLa）形成无序的非极性固体球体。用软琼脂测定HNVEC细胞对DNA损伤有正常的细胞反应，没有转化特性。HNVEC表达上皮标志物细胞角蛋白14 （CK14）和p63，但不表达细胞角蛋白18 （CK18）。接下来，我们利用气液界面（ALI）培养重建hnvec来源的三维阴道上皮。三维阴道上皮具有基底层和根尖层，上皮标记物表达为其起源的人类阴道组织。最后，我们基于重建的三维阴道上皮建立了HSV-2感染模型。在重建的三维阴道上皮的根尖层接种HSV-2 （G株）后，我们观察到明显的病理效应逐渐从根尖层扩散到基底层，并表达了一种病毒蛋白。因此，我们建立了体外二维和三维HSV-2感染模型，可用于HSV-2病毒学和抗病毒药物的发现。
Herpes simplex virus type 2 (HSV-2) infects human genital mucosa and establishes life-long latent infection. It is unmet need to establish a human cell-based microphysiological system for virus biology and anti-viral drug discovery. One of barriers is lacking of culture system of normal epithelial cells in vitro over decades. In this study, we established human normal vaginal epithelial cell (HNVEC) culture using co-culture system. HNVEC cells were then propagated rapidly and stably in a defined culture condition. HNVEC cells exhibited a normal diploid karyotype and formed the well-defined and polarized spheres in matrigel three-dimension (3D) culture, while malignant cells (HeLa) formed disorganized and nonpolar solid spheres. HNVEC cells had a normal cellular response to DNA damage and had no transforming property using soft agar assays. HNVEC expressed epithelial marker cytokeratin 14 (CK14) and p63, but not cytokeratin 18 (CK18). Next, we reconstructed HNVEC-derived 3D vaginal epithelium using air-liquid interface (ALI) culture. This 3D vaginal epithelium has the basal and apical layers with expression of epithelial markers as its originated human vaginal tissue. Finally, we established an HSV-2 infection model based on the reconstructed 3D vaginal epithelium. After inoculation of HSV-2 (G strain) at apical layer of the reconstructed 3D vaginal epithelium, we observed obvious pathological effects gradually spreading from the apical layer to basal layer with expression of a viral protein. Thus, we established an ex vivo 2D and 3D HSV-2 infection model that can be used for HSV-2 virology and anti-viral drug discovery.