全反式维甲酸诱导的Cx43基因在RB细胞中的过表达及其对RB生长的抑制作用  

All trans retinoic acid-induced overexpression of Cx43 gene in RB cells and its inhibition on the growth of RB

作　　者：李立[1] 唐罗生[2] 

机构地区：[1]天津市眼科医院、天津医科大学眼科临床学院、天津市眼科研究所、天津市眼科学与视觉科学重点实验室,300020 [2]中南大学湘雅二医院眼科,长沙410011

出　　处：《中华实验眼科杂志》2014年第10期910-915,共6页Chinese Journal Of Experimental Ophthalmology

摘　　要：背景诱导细胞间隙连接蛋白（＆）基因在瘤细胞中过表达是全反式维甲酸（ATRA）抑制肿瘤细胞生长的重要机制。我们先前的研究已证实，ATRA抑制视网膜母细胞瘤（RB）细胞增生、分化作用的机制与诱导Cx43过表达有关，但药物作用位点及其对在体肿瘤组织生长的影响尚未明确。目的研究ATRA对RB细胞中Cx43基因及其蛋白表达的影响，探讨其作用机制。方法用无水乙醇将ATRA溶解并配制成浓度为1×10^-2mol／L的溶液，使用前再用细胞培养液配制成不同浓度ATRA液。用含体积分数10％热灭活胎牛血清（FBS）的RPMI-1640培养液常规培养人RB细胞株HXO—RB44，将1×10^5-×10“和1×10^-2mol／L的ATRA分别加至培养基中，分别于添加后2、4、6d采集细胞，分别采用Westernblot法和逆转录PCR法检测细胞中Cx43蛋白及mRNA的表达变化。选取15只BALB／c裸鼠，将RB细胞悬液1恤l（含5×10^5个细胞）进行裸鼠右眼前房注射，建立裸鼠眼前房RB模型。将造模成功的11只裸鼠随机分为空白对照组、阴性对照组和1X10。mol／LATRA组，阴性对照组和1×10^-5mol／LATRA组裸鼠分别行0．5％无水乙醇的生理盐水或1×10^-5mol／LATRA前房注射，每3天1次，共注射3周，裂隙灯显微镜下观察各组裸鼠肿瘤生长情况；实验结束时摘除实验眼，用苏木精-伊红染色法进行组织病理学检查。结果Westernblot法检测结果显示，随ATRA作用时间的延长，各组Cx43蛋白表达量（Acx43／AGPDH）均逐渐增加，总体比较差异有统计学意义（F时间=71．31，P=0．00；F分组7．66，P=0．00）；药物作用后2d1×10^-5mol／LATRA组、作用后4d1×10^-6mol／LATRA组、作用后6d1×10^-7mol／LATRA组Cx43蛋白表达量均明显高于空白对照组，差异均有统计学意义（t=3．34，P〈0．叭；t=2．33，P〈0．05；t=3．12，P〈0．01）。逆转录PCR检测结果显示，随ATRA作用时间的延长，各组Cx43 mBackground One of the important machanisms of all trans retinoic acid （ATRA） is to regulate the expression of connexin （Cx） gene. ATRA inhibits the proliferation and differentiation of retinoblastoma （RB） cells,which is related to Cx43. However,the control site of ATRA and its effect on RB tumor in vivo have not beenidentified. Objective This study was to investigate the effect of ATRA on Cx43 expression in RB cells and its approach mechanisms. Methods ATRA solution of 1 × 10^-2 mol/L was prepared with ethanol and formulated into 1 × 10-s, 1 x lO-6and 1× 10^-7 mol/L of solution with culture medium further. Human RB cell line （HXO-RB44） was cultured and treated with different concentrations of ATRA for 2,4 and 6 days, respectively. The expressions of Cx43 protein and mRNA in RB cells were detected by Western blot and reverse transcription PCR （RT-PCR） ,respectively. RB models were established by injecting HXO-RB44 cell suspension into anterior chamber in the right eyes of 15 athymic mice. Eleven successful models were divided into the blank control group, negative control group and 1 × 10^-5 mol/L ATRA group, and 0.5 % normal saline solution with athymic or 1 ×10^5- mol/L ATRA solution was injected into the anterior chamber in the negative control group and 1 × 10^-5 mol/L ATRA group in the 3-day interval for 3 weeks. The model eyes were examined under the slit lamp microscope. The eyeballs were extracted at the end of the experiment for hematoxylin and eosin staining. Results Western blot assay showed that the absorbance values of Cx43 protein （Acx43/AGApDH ） were increased gradually as time lapse of ATRA treatment among the groups （Ftime= 71.31 ,P = 0. 00; Fgroup = 7.66, P = 0.00）. The expressions of Cx43 protein were significantly higher in the 1 × 10^-5 mol/L ATRA group after 2 days, 1 × 10^-6 mol/L ATRA group after 4 days, 1 ×10^-7 mol/L ATRA group after 6 days than those in the blank control group at various time points （ t = 3.34, P〈0.01 ; t = 2.33, P〈0.05 ; t = 3.

关 键 词：全反式维甲酸 视网膜母细胞瘤 细胞间隙连接 动物模型 

分 类 号：R739.7[医药卫生—肿瘤]