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目的 探讨抗凋亡基因 bcl- 2表达对体外培养的葡萄膜黑色素瘤 (uveal melanoma,UM)细胞存活的影响。 方法 通过阳离子脂质体将 bcl- 2反义寡核苷酸 (antisense oligodeoxynucleotides,AS-ODN)转导原代培养 UM细胞 ,应用噻唑蓝 (3,- 4 ,5 Dimethyliazol - 2 ,5 diphenyl tetrazolium bromide,MTT)法测定 bcl- 2 AS- ODN对肿瘤细胞增生的抑制作用 ;提取肿瘤细胞 DNA,检测肿瘤细胞的凋亡情况 ;利用逆转录聚合酶链式反应 (reverse transcription polymerase chain reaction,RT- PCR)和蛋白印迹(Westernblot)技术 ,从 RNA及蛋白水平测定 bcl- 2 AS- ODN对肿瘤细胞 bcl- 2表达的影响。 结果 bcl-2 AS- ODN对 UM细胞的增生有明显抑制作用 ,并且其抑制作用随浓度的增高而增强。bcl- 2 AS- ODN在m RNA及蛋白水平对 bcl- 2基因表达产生明显抑制作用 ,而正义寡核苷酸 (sense oligodeoxynucleotides,S-ODN)对肿瘤细胞的增生则无明显的抑制作用。 结论 bcl- 2 AS- ODN在转录和翻译水平下调 bcl- 2基因表达 ,抑制 UM细胞增生并诱导其凋亡。
Objective To investigate the effect of anti-apoptotic gene bcl-2 on the survival of uveal melanoma (UM) cells cultured in vitro. Methods Primary cultured UM cells were transfected with bcl-2 antisense oligodeoxynucleotides (AS-ODNs) by cationic liposomes. Thiazolyl blue (3, - 4, 5 Dimethyliazol - 2, 5 diphenyl tetrazolium bromide MTT assay was used to determine the inhibitory effect of bcl-2 AS-ODN on the proliferation of tumor cells. The DNA of tumor cells was extracted to detect the apoptosis of tumor cells. Reverse transcription polymerase chain reaction (RT-PCR) ) And Western blotting were used to determine the effect of bcl-2 AS-ODN on the expression of bcl-2 in tumor cells from RNA and protein levels. Results bcl-2 AS-ODN significantly inhibited the proliferation of UM cells, and its inhibitory effect increased with increasing concentration. Bcl-2 AS-ODN significantly inhibited the expression of bcl-2 gene in m RNA and protein levels, whereas sense oligodeoxynucleotides (S-ODN) had no obvious inhibitory effect on the proliferation of tumor cells. Conclusion bcl-2 AS-ODN down-regulates the expression of bcl-2 gene at transcription and translation level, inhibits the proliferation of UM cells and induces apoptosis.