【作者】 姚德生； 李力； Kenneth Garson； Barbara C．Vanderhyden；

【Author】 YAO De-Sheng Li Li Kenneth Garson et al. Department of Gynecological Oncology, Affiliated cancer hospital of Guangxi Medical University, Nanning 530021, China

【机构】 广西医科大学附属肿瘤医院妇瘤科； 加拿大渥太华大学癌症中心；

【摘要】 目的:为了探讨癌基因c—myc和K—ras在卵巢癌发生发展中的作用,以及重组Moloney逆转录病毒作为基因转移的载体的可行性和优点。方法:分离收集CD1小鼠卵巢上皮细胞(MOSE),进行原代培养,建立了MOSE细胞株,将正常的c—myc基因和突变的K—ras基因用Moloney逆转录病毒载体先后转导入MOSE,建立表达c—myc基因和／或 K—ras的细胞系,分别称为Ras细胞、Myc细胞、RM细胞株。我们通过细胞增殖试验、软琼脂克隆形成试验、Matrigel 体外侵袭试验以及裸鼠体内成瘤试验研究转基因后MOSE细胞生物学特性的改变和恶性转化。结果:用携带有c-myc和 K-ras基因的重组逆转录病毒感染MOSE后,用RT-PCR 能够检测到在靶细胞内分别有相应的c—myc或／和K—ras mRNA转录,Western blot能够检测到相应的c—myc (62kDa)或／和K—ras(21kDa)蛋白的表达;细胞增殖实验显示,转导了K—ras的的MOSE细胞(Ras组和RM组)增殖显著快干未转基因的MOSE和Myc(单独c—myc)组(P< 0．01),RM组细胞显著快于Myc组(P<0．05);软琼脂克隆形成试验显示Ras和RM组在软琼脂培养中均能形成克隆集落,而MOSE组和Myc组却不能形成克隆集落;Matrigel 体外侵袭试验显示Ras和RM组能够穿透Matrigel,具有侵袭能力,而MOSE组和Myc组细胞不能穿过Matrigel,不具有侵袭能力;异体移植瘤试验显示,将Ras和RM组细胞腹腔注射于裸鼠体内,有肿瘤形成,而MOSE组和Myc组没有肿瘤形成;免疫组化染色显示,虽然经过长时间体外的生长,形成的肿瘤组织细胞仍然可见有K—ras和c—myc蛋白的表达。结论:重组的Moloney逆转录病毒载体作为转基因的工具,具有高转导效率,可以感染正常的细胞,同时能将目的基因整合到宿主细胞的基因组中,达到稳定表达, 其表达的蛋白质在异体移植瘤的组织中仍然有稳定表达;突变的K—ras可使MOSE发生恶性转化,在体内形成肿瘤; c—myc作为一个核转录调节因子,单独不能使细胞增殖加快,也不能使MOSE发生恶性转化,在体内也不能形成肿瘤,但可协同其他因子如K—ras,加强其功能,赋予细胞更强的生长和侵袭能力。更多还原

【Abstract】 Objective: In order to study the function of c-myc and K-ras in tumongenesis of ovarian cancer, also the reliability of retroviral vector for efficient gene transduction. Methods: Mouse ovarian surface epithelium cells(MOSE) were collected and cultured from the CD1 mice, K-ras or/and c-myc were introduced to MOSE by recombinant Moloney retroviral vector, the phenotypic characteristic of the transgenic MOSE and tumorigenesis were studied by cell proliferation assays, soft-agarose cloning formations, Matrigel invasion assays and tumorige-nicity assays in nude mice. Results: K-ras an c-myc can be easy delivered to the normal MOSE cells by the recombinant retroviruses, mRN A and protein of the target genes can be detected by RT-PCR and Western blot, Cell proliferation assays showed that Ras cell(MOSE-Ras), and RM cell (MOSE-RM) lines growed fast comparing parent cells (MOSE) and Myc(MOSE-Myc) cells(P<0.01), RM cells growed fast than Ras cells(P<0.05), Cell cloning formation assay showed that Ras and RM cells can form colonies in soft-agarose culture, but not in Myc cells and the control (MOSE), Matrigel invasion assay showed that Ras and RM cells have invasion ability, but not in Myc cells and the control (MOSE), Xenograft experiments showed Ras and RM cells can make tumor after intra-peritoneal injected to nude mice, but not in Myc cells and MOSE, Immunohistochemi-cal staining showed the xenograft tumor tissue also expressed the c-myc and K-ras protein after a long term growed. Conclusion: The recombinant Moloney retroviruses of transgene system have high infection efficiency, it can infected the normal cells, the target gene DNA can be integrated to the genome of host cells, stablely expressed target protein; K-ras have the ability of tumorigenesis, it can drive the normal MOSE cells into malignant tumor in nude mice, c-myc acted as a a nuclear transcription factor, can’ t make tumor in vivo, but can co-operate with K-ras.更多还原