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目地:探讨尿激酶型纤溶酶原激活因子在卵巢上皮癌浸润转移中的作用机制。方法:用RT-PCR技术从人卵巢上皮癌组织总RNA中逆转录uPA基因cDNA全长,克隆至pGEM-T Easy Vector,鉴定后与真核表达载体PCMV-HA连接,酶切及测序。用脂质体法将重组质粒DNA转染至SKOV3细胞。加压筛选并培养PCMV-HA-uPA及对照细胞。分别用RT-PCR和W estern blot方法检测转染前后SKOV3细胞uPA的表达。细胞增殖能力测定用四甲基偶氮唑蓝(MTT)法和集落形成实验,细胞周期测定用流式细胞仪法,细胞体外侵袭,迁移和黏附能力测定分别采用Matrigel Invasion,TranswellM igration和Adhersion Assay方法。结果:(1)uPA阳性表达能介导SKOV3细胞克隆形成,与对照组细胞的差异有统计学意义(P<0.05);(2)uPA阳性表达能介导SKOV3细胞的细胞周期中S期比例增加,与对照组细胞的差异有统计学意义(P<0.05);(3)uPA阳性表达介导SK-OV3细胞的体外侵袭,迁移和黏附能力均明显强于对照细胞株,差异有统计学意义(P=0.0002,<0.0001和0.0049)。结论:uPA通过促进肿瘤细胞侵袭,迁移和黏附能力在卵巢上皮癌浸润转移中起了重要作用。
Objective: To investigate the role of urokinase-type plasminogen activator in the invasion and metastasis of epithelial ovarian cancer. Methods: The full length cDNA of uPA gene was reverse transcribed from total RNA of human ovarian epithelial carcinoma tissue by RT-PCR and cloned into pGEM-T Easy Vector. After identification, the full length cDNA of uPA gene was ligated with the eukaryotic expression vector PCMV-HA, digested and sequenced. Recombinant plasmid DNA was transfected into SKOV3 cells by liposome method. The PCMV-HA-uPA and control cells were screened and cultured under pressure. The expression of uPA in SKOV3 cells before and after transfection was detected by RT-PCR and Western blot respectively. Cell proliferation assay MTT assay and colony formation assay were used. Cell cycle assay was performed by flow cytometry, in vitro invasion, migration and adhesion assay. Matrigel Invasion, Transwell Migration and Adhersion Assay method. Results: (1) The positive expression of uPA could mediate the formation of SKOV3 cell clone with statistical significance (P <0.05). (2) The positive expression of uPA could mediate the proportion of S phase in SKOV3 cell cycle (P <0.05). (3) The invasiveness, migration and adhesion of SK-OV3 cells in vitro were significantly enhanced by uPA positive expression compared with the control cell lines, the difference was statistically significant Significance (P = 0.0002, <0.0001 and 0.0049). Conclusion: uPA plays an important role in the invasion and metastasis of epithelial ovarian cancer by promoting tumor cell invasion, migration and adhesion.