THY1基因对上皮性卵巢癌细胞SKOV3生长的抑制作用

Growth inhibitory effects of THY1 gene on epithelial ovarian cancer SKOV3 cells

目的构建THY1真核表达质粒,并探讨THY1基因对上皮性卵巢癌细胞系SKOV3生长的影响。方法通过RT-PCR方法,从人正常卵巢组织中获得THY1基因,将其插入真核表达质粒pcDNA3.1(+)中,构建成重组质粒pcDNA3.1(+)-THY1,并转入大肠杆菌JM109,筛选出含有正确插入片段的克隆,经PCR、酶切及DNA测序鉴定;脂质体介导法转染SKOV3细胞并筛选稳定表达(SKOV3-THY1组),同时设空质粒转染组(SKOV3-Null组)和空白对照组(SKOV3组),RT-PCR和Westernblotting鉴定重组质粒的表达情况;MTT法和流式细胞术检测THY1对SKOV3细胞生长和凋亡等生物学行为的影响。结果经过PCR、酶切及DNA测序证实,外源性THY1基因正确插入到真核表达质粒pcD-NA3.1(+)中,RT-PCR和Westernblotting证实此重组质粒已整合于SKOV3细胞并获稳定表达;MTT法结果提示SKOV3-THY1组的细胞抑制率(第5天的细胞抑制率为56.6%)明显高于SKOV3-Null组(12.5%)(P<0.05);流式细胞术检测结果显示,SKOV3-THY1组凋亡率(31.8%)明显高于SKOV3-Null组(10.5%)和SKOV3组(9.8%),差异有统计学意义(P<0.05),SKOV3-Null组和SKOV3组间差异无统计学意义(P>0.05)。结论成功构建了THY1真核表达质粒pcDNA3.1(+)-THY1,该质粒转染SKOV3细胞可抑制其生长,THY1基因可能在上皮性卵巢癌发生、发展的过程中起重要作用。

Objective To construct THY1 eukaryotic expression plasmid and study its effects on the growth of epithelial ovarian cancer cell line SKOV3. Methods THY1 gene fragment was obtained from normal human ovarian tissue using RT-PCR, and inserted into the eukaryotic expression plasmid pcDNA3.1 （＋） to construct the recombinant plasmid pcDNA3.1 （＋）-THY 1, which was transformed into E. coil JMI09 followed by selection of the positive clones containing the target inserts. The eukaryotic expression plasmid was analyzed by PCR, restriction endonucleases digestion and DNA sequencing. SKOV3 cells divided into SKOV3-THY1, SKOV-3-Null and SKOV3 groups were transfected v/a liposome with the recombinant plasmid pcDNA3.1 （＋）-THY1, empty plasmid, or not transfected, respectively. The expression of THY1 mRNA and its protein were examined by RT-PCR and Western blot methods. The cell growth and apoptosis were evaluated by MTT assay and flow cytometry. Results The gene fragment of exogenous THY1 was correctly inserted into the eukaryotic expression plasmid pcDNA3.1 （＋） as verified by PCR, restriction endonucleases digestion and DNA sequencing, and recombinant expression plasmid pcDNA3.1 （＋）-THY1 transfection resulted in stable expression in SKOV3 cells as shown by RT-PCR and Western blotting. The cell growth inhibition rate of SKOV3-THY1 group （56.6% at the fifth day） was significantly higher than that of the SKOV3-Nu11 group （12.5%, P〈0.05）, and the cell apoptosis rate in SKOV3-THY1 group （31.8%） was significantly higher than those in SKOV3-Null group （10.5%） and SKOV3 group （9.8%, P〈0.05）, but the apoptosis rate between the latter two groups was similar （P〉0.05）. Conclusions The recombinant plasmid pcDNA3. 1（＋）-THY1 can be expressed stably in human ovarian cancer cell line SKOV3. THY1 transfection can inhibit the growth of SKOV3 cells in vitro, suggesting the important role of THY1 gene in pathogenesis and development of ovarian cancer.