[人KIAA1173基因的克隆及转染6-10B细胞的生物学特性]。

San-quan Zhang, Hong Peng, Hui-yong Jiang, Hai Hu, Jin-hua Zhang, Kai-tai Yao, Tong Zhao
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引用次数: 0

摘要

目的:建立稳定表达KIAA1173基因的6-10B细胞系,并研究其生物学行为。方法:从正常骨骼肌组织中提取总RNA,采用RT-PCR方法克隆KIAA1173基因,并将其导入pcDNA3.1(+)载体中。构建重组真核表达载体pcDNA 3.1(+)-KIAA1173,经酶切和测序鉴定,以空载体为对照,脂质体转染6-10B细胞。通过G418筛选获得阳性细胞克隆。采用RT-PCR、原位杂交、免疫细胞化学等方法检测KIAA1173基因在转染后的6-10B细胞中的稳定表达,并采用裸鼠MTT法、细胞侵袭试验和肿瘤发生试验观察转染后细胞的生物学行为。结果:KIAA1173在转染的6-10B细胞中mRNA和蛋白水平均有高表达。与转染pcDNA3.1(+)载体的细胞相比,转染KIAA1173的细胞在裸鼠体内的增殖能力、侵袭能力和致瘤能力均降低。结论:KIAA1173基因在体外和体内均可能具有潜在的鼻咽癌抑癌作用。获得了表达KIAA1173的6-10B细胞系,为进一步研究KIAA1173基因提供了依据。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
[Cloning of human KIAA1173 gene and biological characterization of transfected 6-10B cells].

Objective: To establish a 6-10B cell line with stable expression of KIAA1173 gene and study the biological behaviors of the cells.

Methods: The total RNA was extracted from normal skeletal muscular tissues for cloning of KIAA1173 gene by means of RT-PCR which was subsequently introduced into pcDNA3.1 (+) vector. The recombinant eukaryotic expression vector pcDNA 3.1(+)-KIAA1173 was constructed and identified by endonuclease digestion and sequencing before transfection into 6-10B cells via lipofectamine with the empty vector as the control. The positive cell clones were obtained by G418 selection. Stable expression of KIAA1173 gene in the transfected 6-10B cells was determined by RT-PCR, in situ hybridization and immunocytochemistry, and the biological behaviors of the transfected cells were observed by MTT assay, cell invasion assay and tumorigenesis assay in nude mice.

Results: High expression of KIAA1173 at both mRNA and protein levels was observed in the transfected 6-10B cells. The capability of proliferation, invasion and tumorgenicity of the KIAA1173-transfected cells in nude mice was lowered in comparison with those of the cells transfected with pcDNA3.1 (+) vector (P<0.05).

Conclusions: KIAA1173 genes may function as a potential tumor suppressor of nasopharyngeal carcinoma both in vitro and in vivo. The 6-10B cell line expressing KIAA1173 has been obtained, which can be helpful for further study of KIAA1173 gene.

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