反义MAT1重组腺病毒对人胰腺癌细胞BxPC-3的周期调控作用

目的探讨MAT1基因在胰腺癌BxPC3细胞周期G1/S转换中的作用。方法应用同源重组技术构建含反义MAT1基因的腺病毒载体;细胞生长分析采用台盼蓝染色计数法;Western印迹检测细胞MAT1、细胞周期素D1(cyclinD1)、cyclinE和pRb的蛋白表达;采用流式细胞仪分析反义MAT1基因转染后BxPC3细胞周期的变化。结果编码反义MAT1的重组腺病毒Ad MAT1AS感染滴度为5×1010pfu/ml;BxPC3细胞感染Ad MAT1AS后,MAT1、cyclinE和pRb蛋白表达明显下调,同时出现了明显的细胞G1期阻滞,79%的细胞处于G0/G1期,而空白对照组及空载体组则为40%至44%的细胞处于G0/G1期。结论MAT1基因在胰腺癌BxPC3细胞周期G1→S转换中发挥重要作用。

The effect of recombinant adenovirus encoding antisense MAT1 on cell cycle of human pancreatic cancer cells BxPC-3

OBJECTIVE: To investigate the regulatory effect of the human MAT1 gene on the cell cycle G(1)/S transition of human pancreatic cancer BxPC-3 cells. METHODS: To construct the replication deficient recombinant adenovirus of antisense MAT1 gene using homologous recombination by AdEasy system. Cell growth assay was carried out by counting alive cells after trypan blue exclusion. The protein expressions of MAT1, cyclin D1, cyclin E, and Rb were detected by western blotting. The cell cycle status was determined by flow cytometry. RESULTS: The recombinant adenovirus encoding antisense MAT1 fragment Ad-MAT1AS was obtained with the titer of expression was 5 x 10(10) pfu/ml. The expression of MAT1 of BxPC-3 was significantly reduced after Ad-MAT1AS infection. In this case BxPC-3 cell cycle was arrested in G(1) phase. The estimated proportion of G(0)/G(1) phase cells in the control for blank and vector cultures ranged from 40% to 44%. In contrast, 79% of the Ad-MAT1AS cells were in G(0)/G(1) phase. Cyclin E and pRb gene expression changes were observed in the infected cells. CONCLUSION: The results suggest that MAT1 gene may play an important role in the regulation of cell cycle G(1)-->S transition of BxPC-3 cells.