Ad-NLS-RARα对HL-60细胞增殖及ATRA诱导的HL-60细胞分化的影响及其机制

Effect of Recombinant Adenovirus Carrying NLS-RARαGene on the Proliferation of HL-60 Cell and the Differentiation of HL-60 Cells Induced by ATRA and Relevant Mechanism

目的将已构建验证成功的重组腺病毒Ad—NL＆RARα感染至HL-60细胞中，探讨其对HL-60细胞增殖及全反式维甲酸（ATRA）诱导的HL-60细胞分化的影响及机制。方法将验证正确的腺病毒经Protaminesulfate辅助感染Hl-60细胞，检测感染效率，用RT-PCR和Westernblot检测目的基因在HL-60细胞中的表达情况。MTT法检测细胞增殖情况。病毒感染HL-60细胞24h后，用2．5μmol／LATRA处理，流式细胞术（FCM）检测细胞表面分化抗原CDllb的表达。RT—PCR和Western blot检测GMYC基因在mRNA和蛋白水平的表达。结果在Protamine sulfate辅助感染作用下，重组腺病毒Ad—NLS-RARα和阴性对照腺病毒Ad—KZ对HL-60细胞的感染效率可达70％～80％。NL＆RARα基因的RT—PCR和Westernblot结果显示，感染了重组腺病毒Ad—NL＆RAR口的HL-60细胞的NLS-RAR口基因的mRNA及蛋白表达水平明显增高（P〈0．05）。MTT法检测表明感染了重组腺病毒Ad-NLS-RARα的细胞增殖能力增高（P〈O．05）。FCM检测结果显示，经ATRA处理后，感染Ad—NLS-RAR口重组腺病毒的HL-60细胞，CDllb表达较阴性对照组和未感染组下调（P〈O．05）。C-MYC基因的RT—PCR和Westernblot检测结果表明，经ATRA处理后，感染Ad—NLS-RARα重组腺病毒的细胞C—MYC基因的mRNA和蛋白水平均高于其他两组（P〈0．05）。结论重组腺病毒Ad—NL-SRARα可以促进HL-60细胞的增殖，并通过上调GMYC基因的表达，抑制ATRA诱导的HL-60细胞分化。

Objective To explore the effect and mechanism of recombined adenovirus carrying NLS-RARα gene on proliferation of HL-60 cells and the differentiation of HL-60 cells induced by ATRA. Methods HL-60 cells was infected with Ad-NLS-RARα and control virus Ad-KZ. The efficiency of infection was detected by FCM. The mRNA and protein levels of NLS-RARα were assessed by Real-time PCR （RT-PCR） and Western blot, respectively. MTT assay were applied to determine proliferation of HL-60 cells. Cell surface differentiation antigen CDllb of infected HL-60 cell induced by ATRA was examined by FCM. The mRNA and protein levels of C-MYC of infected HL-60 cell induced by ATRA were determined by Real-time PCR （RT PCR） and Western blot assay. Results The efficiency of infection of Ad-NLS-RARα and Ad-KZ on HL-60 cell was 70~/oo-80%. The mRNA and protein levels of NLS-RARct gene of HL-60 cells which infected with Ad-NLS-RARα were both obviously higher than that of the cells which infected with Ad-KZ and non-infected （P〈0.05）. The proliferation ability of HL-60 cell infected with Ad-NLS-RARct was significantly increased （P〈0.05）. The level of CDllb of HL-60 cell infected with Ad-NLS-RARα and induced by ATRA was clearly decreased than control groups （P〈0.05）. The mRNA and protein levels of C-MYC gene of HL-60 cells infected with Ad-NLS-RARα and induced by ATRA were both obviously higher than that of the cells which infected with A＆KZ and non-infected （P〈0.05）. Conclusion The recombined adenovirus Ad-NLS-RARα can increase the proliferation ability of HL-60 cell, and inhibit the differentiation of HL-60 cell through reduce the expression level of C-MYC gene.