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目的建立稳定表达防御素mBD2的恶性黑色素瘤细胞B16-mBD2,研究mBD2对B16细胞生物学特性的影响。方法在前期构建好mBD2真核表达质粒的基础上,脂质体法转染恶性黑色素瘤B16细胞,G418筛选得到稳定表达细胞株B16-mBD2,RT-PCR和Western blot从mRNA和蛋白角度鉴定mBD2分子的表达;MTT法鉴定其生长曲线;流式细胞仪分析细胞周期分布、凋亡情况及细胞膜表面分子CD80、CD86、MHCⅠ(H-2Kd)、MHCⅡ(I-Ad)的表达。结果成功构建稳定表达mBD2的B16细胞,RT-PCR和Western blot分别从mRNA及蛋白水平验证了mBD2分子表达;稳定表达mBD2的细胞B16-mBD2与转染空载体的B16-p细胞及野生型B16细胞相比,细胞增殖明显减慢,从48 h开始,各时间点细胞活力显著降低(P<0.05);B16-mBD2细胞处于S期的比例(36.03±0.97)%显著增高(P<0.05);凋亡率及细胞膜表面分子CD80、CD86、MHCⅠ(H-2Kd)、MHCⅡ(I-Ad)表达无明显差异(P>0.05)。结论 mBD2明显抑制恶性黑色素瘤B16细胞的增殖,其机制可能与诱导细胞周期S期阻滞有关;mBD2对细胞凋亡率及细胞膜表面免疫分子的表达无影响。
Objective To establish B16-mBD2 cells stably expressing defensins mBD2 and study the effect of mBD2 on the biological characteristics of B16 cells. Methods The mBD2 eukaryotic expression plasmid was constructed and transfected into B16 cells by lipofectamine 2000. The stable cell line B16-mBD2 was selected by G418 screening. The expression of mBD2 mRNA and protein was identified by RT-PCR and Western blot. The expression of CD80, CD86, MHCⅠ (MHCⅡ) and MHCⅡ (I-Ad) were detected by MTT assay. Cell cycle distribution and apoptosis were analyzed by flow cytometry. Results B16 cells stably expressing mBD2 were successfully constructed. The expression of mBD2 was verified by RT-PCR and Western blot respectively. The expression of mBD2 was detected by RT-PCR and Western blotting. The expression of mBD2 in B16-mBD2 cells stably expressing mBD2 and B16-p cells transfected with empty vector and wild type B16 (P <0.05). The proportion of B16-mBD2 cells in S phase (36.03 ± 0.97)% was significantly increased (P <0.05) There was no significant difference in apoptotic rate and expression of CD80, CD86, MHCⅠ (H-2Kd) and MHCⅡ (I-Ad) on the cell membrane (P> 0.05). Conclusion mBD2 significantly inhibits the proliferation of B16 melanoma cells. The mechanism may be related to the induction of cell cycle S phase arrest. MBD2 has no effect on the apoptosis rate and the expression of cell surface immune molecules.