通过 SV40 大 T 抗原转染构建和鉴定人类结直肠腺瘤上皮细胞系

Xiangxue Meng, Xinyue Han, Xiangying Lin, Guanhong Li, Jingnan Wang, Ao Sun, Xiaochen Fu, Bowen Xu, Donghua Yang, Yanping Wu, Min Zhang, Xiaoling Fu
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引用次数: 0

摘要

背景:大肠腺瘤是大肠癌发展的关键一步。建立永生化的人源大肠腺瘤上皮细胞系将为研究癌前病变机制、筛选新型药物疗效和构建体内疾病模型提供工具。目的:本研究旨在通过SV40-LT抗原基因转染建立天然低表达LHPP癌前病变上皮细胞系:方法:将 Simian vacuolating virus 40(SV40)、SV40-LT 过表达慢病毒载体转染原代人大肠腺瘤性息肉上皮细胞。对转染的细胞进行筛选,并对筛选出的细胞进行扩增,得到上皮细胞系:IHCRA- CELL。通过形态观察、细胞增殖、定量实时 PCR(qPCR)和短串联重复序列(STR)实验对细胞进行鉴定。从形态上看,细胞表现出上皮样特征,如多边形、脱膜线粒体和强阳性角蛋白染色。转染细胞与原代细胞无明显差异。通过 STR 鉴定实验,在细胞系检索中未发现匹配的细胞系:通过SV40-LT抗原基因转染,我们成功建立了一个天然的低表达LHPP癌前上皮细胞系,该细胞系已申请专利,现保存在中国典型培养物保藏中心。实验证实,转化后的细胞保持了上皮细胞的表型和生物学特性。该细胞系可用于研究癌前病变的机制、筛选新型药物的疗效以及构建体内疾病模型。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Construction and Identification of a Human Colorectal Adenoma Epithelial Cell Line by SV40 Large T-Antigen Transfection.

Background: Colorectal adenoma represents the critical step in the development of colorectal cancer. The establishment of an immortalized epithelial cell line of colorectal adenoma of human origin would provide a tool for studying the mechanism of precancerous lesions, screening the efficacy of novel drugs, and constructing in vivo disease models. Currently, there is no commercially available stable supply of epithelial cells from precancerous lesions.

Aims: This study aimed to establish a natural LHPP low-expressing precancerous epithelial cell line by SV40-LT antigen gene transfection.

Methods: Simian vacuolating virus 40(SV40), SV40-LT overexpressed lentivirus vector, was transfected into primary human colorectal adenomatous polyp epithelial cells. The transfected cells were screened, and the screened cells were amplified to obtain the epithelial cell line: IHCRA- CELL. The cells were identified by morphological observation, cell proliferation, Quantitative real-time PCR (qPCR), and Short Tandem Repeats (STR) experiments. Morphologically, the cells showed epithelial-like characteristics, such as polygon shape, desmosomes mitochondria, and strong positive keratin staining. There was no significant difference between the transfected cells and the primary cells. Through the STR identification experiment, no matching cell lines were found in the cell lines retrieval.

Conclusion: We successfully established a natural LHPP low-expressing precancerous epithelial cell line by SV40-LT antigen gene transfection, which has been patented and is now preserved in the Chinese Typical Culture Preservation Center. It was verified that the transformed cells maintained the phenotype and biological characteristics of epithelial cells. This cell line can be used to study the mechanism of precancerous lesions, screen the efficacy of novel drugs, and construct in vivo disease models.

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