THY1基因真核表达质粒的构建及其对卵巢癌细胞SKOV3生长的影响

Construction of THY1 Eukaryotic Expression Plasmid and Its Effects on Growth of Ovarian Cancer SKOV3 Cells

构建THY1真核表达质粒并转染卵巢癌细胞SKOV3,探讨其对SKOV3细胞生长的影响。通过目的基因克隆,构建pcDNA3.1(+)-THY1质粒并转染SKOV3细胞,实验分3组:重组质粒转染组(SKOV3-THY1)、空质粒转染组(SKOV3-Null)和未转染组(SKOV3)。RT-PCR和免疫细胞化学方法鉴定THY1表达情况;MTT和流式细胞术检测THY1对SKOV3细胞增殖及周期的影响。DNA测序证实THY1正确插入pcDNA3.1(+)中,并整合于SKOV3细胞;SKOV3-THY1组的细胞抑制率明显高于SKOV3-Null组;且其G1期细胞比例明显高于另两组。结论:S期细胞比例明显低于另两组。结论:成功构建THY1真核表达质粒,该质粒可抑制SKOV3细胞生长。

This study was designed to construct THY1 eukaryotic expression plasmid and assess its effects on epithelial ovarian cancer cell line SKOV3. The gene fragment coding for THY1 was inserted into pcDNA3.1 （ ＋ ） for constructing the recombinant plasmid pcDNA3.1 （ ＋ ）-THY1. The eukaryotie expression plasmid was analyzed by PCR, restriction endonucleases digestion and DNA sequencing. The recombinant plasmid pcDNA3.1 （＋）-THY1 was transfected into SKOV3 cells by liposome protocol. The experimental cells were classified into three groups： SKOV3-THY1, SKOV3-Null and SKOV3. The pcDNA3.1 （＋ ）-THY1 has been transfected into SKOV3 cells by RT-PCR and immunohistochemistry. The cell inhibitory rate of SKOV3-THY1 （56.6% at the fifth day） was higher than that of SKOV3-Null （ 12.5%）, there was significant difference between them[P〈0.05）. The ratios of G1 phase of SKOV3 cells after transfection were increased and the ratios of S phase were decreased significantly. There was significant difference between SKOV3-THY1 and SKOV3-Null or SKOV3（P〈0.05）, but there was no significant difference between SKOV3-Null and SKOV3 （P〉0. 05）. We have constructed the recombinant plasmid pcDNA3.1 （＋）-THY1 sucessfully. THY1 transfection can inhibit the growth of SKOV3 cells in vitro.