Xiao Chang-Yi, Fu Bing-Bing, Li Zhi-Ying, Mushtaq Gohar, Kamal Mohammad Amjad, Li Jia-Hua, Tang Gui-Cheng, Xiao Shuo-Shuang
Tumor Research Institute, China Three Gorges University, Yichang, 443002 Hubei Province People's Republic of China.
Department of Gynaecology and Obstetrics, The Second Clinical Medical College of China Three Gorges University, Yichang, 443000 Hubei Province People's Republic of China.
Cancer Cell Int. 2015 May 23;15:53. doi: 10.1186/s12935-015-0206-0. eCollection 2015.
The goal of this study was to identify the nature of the inclusion bodies that have been found in HeLa cells (cervical cancer immortal cell line) by electron microscope and to determine whether the major capsid protein (L1) of human papillomavirus (HPV) can be expressed in HPV-positive uterine cervix cancer cells.
HPV L1 protein expression in HeLa cells was detected with anti-HPV L1 multivalent mice monoclonal antibody and rabbit polyclonal anti-HPV L1 antibody by ELISA, light microscope immunohistochemistry, electron microscope immunocytochemistry and Western blotting assays. Reverse transcriptional PCR (RT-PCR) was performed to detect the transcription of L1 mRNA in HeLa cells. The immortalized human keratinocyte HeCat was used as the negative control.
HPV L1 proteins reacted positively in the lysate of HeLa cells by ELISA assays. HRP labeled light microscope immunohistochemistry assay showed that there was a strong HPV L1 positive reaction in HeLa cells. Under the electron microscope, irregular shaped inclusion bodies, assembled by many small and uniform granules, had been observed in the cytoplasm of some HeLa cells. These granules could be labeled by the colloidal gold carried by HPV L1 antibody. The Western blotting assay showed that there was a L1 reaction strap at 80-85 kDa in the HeLa cell lysates, hence demonstrating the existence of HPV18 L1 in HeLa cells. RT-PCR assay showed that the L1 mRNA was transcribed in HeLa cells.
The inclusion bodies found in the cytoplasm of HeLa cells are composed of HPV18 L1 protein. Since HeLa cell line is a type of cervical cancer cells, this implies that HeLa cells have the ability to express HPV L1 proteins.
本研究的目的是通过电子显微镜鉴定在HeLa细胞(宫颈癌永生细胞系)中发现的包涵体的性质,并确定人乳头瘤病毒(HPV)的主要衣壳蛋白(L1)是否能在HPV阳性的子宫颈癌细胞中表达。
采用抗HPV L1多价小鼠单克隆抗体和兔抗HPV L1多克隆抗体,通过ELISA、光学显微镜免疫组织化学、电子显微镜免疫细胞化学和蛋白质免疫印迹分析检测HeLa细胞中HPV L1蛋白的表达。采用逆转录PCR(RT-PCR)检测HeLa细胞中L1 mRNA的转录情况。将永生化人角质形成细胞HeCat用作阴性对照。
ELISA分析显示,HPV L1蛋白在HeLa细胞裂解物中呈阳性反应。辣根过氧化物酶标记的光学显微镜免疫组织化学分析显示,HeLa细胞中有强烈的HPV L1阳性反应。在电子显微镜下,在一些HeLa细胞的细胞质中观察到由许多小而均匀的颗粒组装而成的不规则形状的包涵体。这些颗粒可用HPV L1抗体携带的胶体金标记。蛋白质免疫印迹分析显示,HeLa细胞裂解物中在80 - 85 kDa处有一条L1反应带,从而证明HeLa细胞中存在HPV18 L1。RT-PCR分析显示,HeLa细胞中L1 mRNA被转录。
在HeLa细胞细胞质中发现的包涵体由HPV18 L1蛋白组成。由于HeLa细胞系是一种宫颈癌细胞,这意味着HeLa细胞具有表达HPV L1蛋白的能力。