THY1基因影响上皮性卵巢癌SKOV3细胞株生长的体内和体外实验研究

Effects of THY1 Gene on Growth of Epithelial Ovarian Cancer SKOV3 Cell Lines in Vitro and in Vivo

目的构建THY1基因真核表达载体,并转染卵巢癌细胞株SKOV3,观察重组载体在体内和体外对SKOV3生长的影响。方法本研究于2005年3月至2007年3月通过目的基因克隆、载体构建技术构建重组载体pcDNA3.1(+)-THY1,转染SKOV3(SKOV3-THY1组),同时设空载体转染组(SKOV3-Null组)和空白对照组(SKOV3组),甲基噻唑基四唑(methyl thiazolyl tetrazolium,MTT)测定法及流式细胞术检测细胞生长及细胞周期变化;并建立雌性裸鼠异体移植卵巢癌模型,观察瘤体生长速率。结果PCR、酶切及DNA测序证实,THY1基因正确插入真核表达载体pcDNA3.1(+),RT-PCR和蛋白质斑迹法(Western blot)证实重组载体已整合于SKOV3;SKOV3-THY1组细胞抑制率明显高于SKOV3-Null组(P<0.05);建立裸鼠致瘤模型4周后,SKOV3-THY1组瘤体体积较SKOV3-Null组和SKOV3组显著降低(P<0.05)。结论THY1真核表达载体能抑制SKOV3的恶性增殖,THY1基因可能在上皮性卵巢癌发生发展中起重要作用。

Objective To construct THY1 gene eukaryotic expression plasmid and study its effects on epithelial ovarian cancer SKOV3 cell lines. Methods The gene fragment code for THY1 was obtained from human normal ovarian tissues using RT-PCR, and inserted into the eukaryotic expression plasmid pcDNA3. 1 （＋） to construct the recombinant plasmid pcDNA3. 1 （＋）-THY1, which was transfected into SKOV3 cells from March 2005 to March 2007. Experimental cells were classified into 3 groups： SKOV3-THY1 group, SKOV3 Null group and SKOV3 group. The expression of THY1 mRNA and its protein were examined by RT-PCR and Western blot. The cell proliferation and cell cycles were evaluated by methyl thiazolyl tetrazolium （MTT）assay and flow cytometry. Tumor sizes were measured in vivo tumorigenicity studies. Results The gene fragment of THY1 was correctly inserted into the eukaryotic expression plasmid pcDNA3. 1 （＋） as verified by PCR, restriction endonucleases digestion and DNA sequencing, and the pcDNA3. 1 （＋）- THY1 has been transfected into SKOV3 cells and obtained stable expression by RT-PCR and Western blot methods. The cell inhibitory rate of the SKOV3-THY1 group （56.6% at the 5th day） was higher than the SKOV3-Null group （12.5 %）, there was significant difference between them （P〈 0. 05）. After transfected by vector pcDNA3. 1 （＋）- THY1, the ratio of G： phase of SKOV3 cells was increased and the ratio of S phase was decreased significantly. There were significant difference between SKOV3- THY1 group and SKOV3-Null group or SKOV3 group （P〈0.05）, but there were no significant difference between SKOV3-Null group and SKOV3 group （P 〉 0. 05）. In vivo tumorigenicity studies, tumors grew quite rapidly in the null transfectants compared with the THY1- transfected cells over the next 4 weeks. Conclusion THY1 transfection can inhibit the growth of epithelial ovarian cancer SKOV3 cells in vitro and in vivo. THY1 gene may play an important role in generation and development of ovarian cancers.