人類非小細胞肺癌 ( NSCLC ) 細胞的生長受到第一型轉形生長因子 ( TGF-α ) 對上皮生長因子受體 ( EGFR ) 的自泌刺激調控。先前研究顯示人類扁平 ( squamous ) 非小細胞肺癌細胞H226的腦轉移細胞株H226Br,由於高度表現EGFR故對外加TGF-α的刺激更為敏感。為了更深入瞭解降低 EGFR表現對細胞生長的影響,本實驗選用人類EGFR cDNA氮端約1.8 kb長 ( 位於EGFR與生長因子結合的部位 ) 的片段,反向插入擁有cytomegalovirus啟動子的pcDNA3質體中,在轉殖細胞中藉由表現的反義RNA中和細胞自身正向的EGFR mRNA,待基因在細胞表現受到抑制後,觀察細胞表型的變化。
本研究以liposome轉殖反義基因質體至細胞中,經neomycin篩選後,得到三個穩定生長的細胞株,實驗方式主要是探討含反義基因質體的細胞與母細胞間的差異,同時也比較含反義基因質體細胞間的不同。
首先以PCR放大NEO基因及EGFR基因,確定經選殖的細胞皆含有抗藥基因,而且證實反義基因質體已經插入基因組DNA中。分析含反義基因質體細胞的生長速率及生長效率,顯示較母細胞及僅含載體細胞降低。此外轉殖反義基因質體也造成細胞外型的改變,例如:外型較母細胞大、細胞群聚間有明顯的界線等。由RT-PCR放大細胞部分EGFR基因的結果,證實EGFR的RNA已受到反義基因的干擾,但是,不同細胞的EGFR受到抑制的程度也不相同。外加上皮生長因子 ( EGF, 10 ng/ml ) 刺激結果,顯示感染反義基因質體細胞的自體磷酸化及增殖作用明顯受到抑制。分析細胞週期分佈發現,感染反義基因質體細胞主要停滯在G2-M期間,這是因為細胞週期調節因子cyclin B1受到抑制或延遲發生所致,因此造成細胞生長速率降低而與母細胞不同。
本研究發現藉由反義上皮生長因子受體的表現,可有效抑制調節因子的表現而影響細胞的生長,這項結果為抑制肺癌細胞生長建立了一個很好的模式,這份初步研究結果,對於腫瘤細胞的抑制,尤其是同型合子p53突變細胞研究提供了一個新的研究方向。
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