【摘要】 目的探讨表皮生长因子受体(EGFR)反义cDNA联合野生型p16β在体外对人喉癌Hep-2细胞信号转导的干预作用。方法将已纯化的EGFR反义cDNA重组腺病毒和p16β在体外转染Hep-2喉癌细胞,采用MTT实验、流式细胞术(FCM)、免疫组化、Westernblot等方法检测Hep-2细胞增殖抑制作用;进行细胞周期、DNA含量、细胞凋亡率的定量分析;检测重组腺病毒对Hep-2细胞EGFR蛋白表达的抑制和p16β蛋白过表达。结果反义EGFRmRNA表达重组腺病毒能有效抑制Hep-2细胞的增殖活性;超过77.7%以上的被转染Hep-2细胞被阻滞在G0/G1期;转染细胞的凋亡率升高;同时出现EGFR蛋白表达的抑制和p16β蛋白的过表达。当p16β重组腺病毒和EGFR反义cDNA重组腺病毒联合转染Hep-2细胞后其抑制Hep-2细胞增殖活性、细胞周期阻滞、抑制细胞调亡作用明细增强。结论p16β和EGFR反义cDNA能有效地干预人喉癌Hep-2细胞的信号转导机制,从而达到抑制Hep-2细胞增殖、诱导细胞凋亡的作用。更多还原

【Abstract】 Objective To test the effect of p16β and EGFR-antisense cDNA in signal transduction on Hep-2 laryngeal squamous cell carcinoma in vitro.Methods The Hep-2 laryngeal squamous carcinoma cells were transfected by recombinant adenovirus AdEasy-EGFR-antisense and AdEasy-p16β in vitro.The inhibition of the EGFR expression and cell growth and changes of cell cycle,DNA content,apoptosis and ultramicrostructure of the Hep-2 cells were examined by MTT,Western blotting analysis,Flow cytometry analysis,Immunohistochemistry,and transmission electron microscope respectively.Results The proliferation of the Hep-2 cells was inhibited significantly by the infection of the Ad-Ad-p16β or Ad-antisense EGFR.The infection also accelerated the apoptosis of the cancer cells.The proportion of cells in G0/G1 phases increased to more than 77.7%.The Ad-antisense EGFR-infected cells showed lower protein expression of EGFR.The P16β protein over expression was observed in the Ad-p16β-infected cells.Conclusion The transfection of Ad-Ad-p16β and Ad-antisense EGFR into Hep-2 cells leads to over-expression of Ad-p16β and under-expression of EGFR,along with G1-phase arrest and apoptotic cell death.Both EGFR and Ad-p16β play important roles in the genesis,growth and differentiation of the human laryngocarcinoma cells.更多还原