摘要
目的本研究通过单独和联合使用9-顺式维甲酸（9-cis retinoic acid，9-cis RA）和1α，25二羟维生素D3[1 alpha，25-dihydroxy vitamin D3，1，25（OH）2D3]，观察其对神经母细胞瘤（neuro—blastoma，NB）细胞系生长、分化、凋亡的影响，探讨其协同作用的效果及相关机制。方法将NB SK—N-SH细胞分为A（对照组）、B（9-cis RA用药组）、C（1，25（OH）2D3用药组），D（9-cis RA和1，25（0H）2D3联合用药组）四组，在处理后适当的时间用四甲基偶氮唑盐（MTT）法检测细胞抑制率，倒差显微镜观察细胞形态的改变，流式细胞仪（flow cytometry，FCM）检测细胞凋亡的变化，同时用逆转录多聚酶链反应（reverse transcription-polymerase chain reaction，RT-PCR）检测维甲酸受体β（retinoic acid receptor beta，RARβ）的mRNA表达情况。结果单独使用9-cis RA或1，25（OH）2D3和联合用药的细胞生长均受到抑制，并呈时间依赖关系，其中联合用药组抑制率明显高于单独用药组（P〈0．05），形态学分化明显，MTT法测定显示联合用药能显著提高NB细胞抑制率并明显优于单独用药组（P〈0．05），FCM钙依赖性磷脂结合蛋白V和7-氨基放线菌素D（AnnexinV／7-AAD）染色法检测显示联合用药对诱导NB细胞早期凋亡和坏死具有显著的协同作用，RT-PCR检测发现联合用药组RARβ的mRNA表达明显高于其他组。结论9-cis RA联合1，25（OH）2D3在体外对NB SK-N-SH细胞系的生长抑制、分化、凋亡能产生明显的协同抑制作用，在小剂量的情况下即能起到显著的治疗效果，明显优于其单独用药，其协同作用的机制可能为通过增加RARβ的mRNA表达水平来抑制肿瘤的生长，为其联合应用于临床提供理论依据和指导。
Objective To investigate the effects of 9-cis retinoic acid （9-cis RA） and 1,25 （OH）2D3 on the growth, differentiation and apoptosis of human neuroblastoma （NB） SK-N-SH cell line in vitro, and explore the synergism and related mechanism. Methods The NB SK-N-SH cells were divided into： Group A as a control , Group B with 9-cis RA ,Group C. with t ,25（OH）2D3 and Group D in combination with 9-cis RA, 1,25（OH）2D3. At day 3,6,9,12 , the cellular morphological changes were observed under microscopy. The growth inhibition rate was measured by MTT assay, the apoptosis was assayed with by flow cytometry, and the expression of RARβ mRNA was determined by reverse transcription-polymerasw chain reaction （RT-PCR）. Results The application of 9-cis RA and 1,25（OH）2D3 alone or in combination, inhibited the growth of SK-N - SH cells in a time-dependent relation. The growth inhibition and morphological differentiation of NB cells were more obvious in Group D （P〈0. 05）. Annexin V/7-AAD staining FCM demonstrated the significant synergistic effects of 9-cis RA and 1,25（OH）2D3 on inducing cell apoptosis. RT-PCR showed that the expression of RARβ mRNA on combination group was higher than the others. Conclusions The combination of 9- cisRA and 1,25 （OH） 2 D3 produces significant synergistic inhibitory effects on the growth, differentiation and apoptosis of NB SK-N-SH cells in vitro with low dosage. The synergistic mechanism could be caused by the expression of RARβ mRNA to inhibit the growth of tumor.
出处
《中华小儿外科杂志》
CSCD
北大核心
2009年第3期151-154,共4页
Chinese Journal of Pediatric Surgery
