子宫内膜腺上皮永生化细胞模型的建立

Establishment of Human Immoratalized Endometrial Glandular Cells Model

目的建立永生化人子宫内膜HPV16型E6和E7基因诱导的腺细胞,以促进对其的细胞生物学、生长分化调节等方面的研究。方法将质粒pcDNA3.1(+)/HPV16E6E7转入腺上皮细胞中,G418筛选,WesternBlot检测目的基因的表达;用相差显微镜观察细胞变化;上机分析细胞周期。结果转化后腺细胞形态无明显改变,相差显微镜下观察细胞贴壁较紧,为多边形或圆形,密集区域呈镶嵌状排列,阳性细胞灶胞浆被均染成棕黄色,胞核呈蓝色;转化后细胞倍增时间约为52.8 h,对数生长期后细胞停止分裂增生,活性降低;转化后的细胞无凋亡峰出现,S期比例为33.23%,明显增加。与未转化细胞相比差异具有统计学意义。结论转染HPV16E6、E7基因至体外培养的子宫内膜腺上皮细胞寿命明延长,得到了可以在体外传代30次以上的转化细胞;具有正常细胞的表型和生长特性,有较强的增殖能力却不具有致瘤性。

【Objective】 To establish immortalized human endometrial adenocyte induced by E6 and E7 genes of human papilloma virus type 16.To study the cytobiology,accommodation of growth and differentiation of adenocyte.【Methods】 Construct and extract recombinant plasmid pcDNA3.1（＋）/HPV16E6E7,then transfer it into primary cultured endometrial glandular epithelial cell.After G418 screening,detect gene expression at the protein level by Western Blot;Observe cell transmutation by phase contrast microscope and analyze cell cycle.【Results】 The morphous of transformant has no significant alteration.They are polygon or round,and have well adherence under phase contrast microscope.The dense area arrange mosaicly.Cytoplasms are stained yellow brown and nucleuses are stained blue.Multiplication time of transformed cell is about 52.8 hours.The cells stop fission and proliferation after logarithmic growth phase.The transformant has no apoptotic peak.S phase fraction was 33.23%,significantly increased.Compared with untransformed cells,the difference has statistical significance.【Conclusion】 The life time of cultured endometrial glandular epithelial cell transfected by HPV16E6,E7 genes is prolonged.The transformant can pass more than 30 times.Transformed cells are similar to normal cells in the phenotype and growth characteristics,but have no tumorigenicity.