人KIAA1173基因的克隆、转染及生物学特性的实验研究

目的建立表达KIAA1173基因的鼻咽癌6-10B细胞模型,并观察转染细胞的生物学活性.方法从新鲜肌肉组织中提取总RNA,采取逆转录PCR得到人KIAA1173基因cDNA.获得的人KIAA1173基因克隆到真核表达载体pcDNA3.1（＋）,进行酶切和序列鉴定.将重组质粒pcDNA3.1（＋）-KIAA1173和空载体利用阳离子脂质体介导转入鼻咽癌6-10B细胞,经G418筛选阳性克隆.用RT-PCR、原位杂交及免疫细胞化学等方法检测KIAA1173基因在6-10B细胞中的稳定表达情况,并通过MTT法、肿瘤细胞侵袭实验及裸鼠体内成瘤性实验方法,检测KIAA1173基因对鼻咽癌细胞的增殖、侵袭及成瘤能力的影响.以空白质粒转染组作为对照.结果在mRNA和蛋白水平证实,转染细胞内有KIAA1173基因的高表达,表明细胞可表达人KIAA1173基因.6-10B在转染了pcDNA3.1（＋）-KIAA1173后,较转染空载体pcDNA3.1（＋）,肿瘤细胞的增殖能力、侵袭能力及裸鼠体内成瘤能力明显降低（P＜0.05）.结论结果提示KIAA1173基因可能是一鼻咽癌肿瘤抑制基因.获得生物学活性稳定的KIAA1173基因高表达的鼻咽癌细胞模型,为进一步研究奠定了基础.

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.