摘要
目的探讨葡萄糖调节蛋白78（GRP78）基因在卵巢上皮性癌（卵巢癌）细胞自噬与凋亡中的作用及其机制，明确其对卵巢癌细胞生长及顺铂敏感性的影响。方法以钙选择性螯合剂——BAPTA-AM、钙离子载体——A23187分别下调、上调卵巢癌细胞系SKOV3细胞中GRP78基因的表达，实验分为3组，BAPTA．AM组：加入BAPTA—AM，终浓度为40μmol／L，作用时间为1h；A23187组：加入A23187，终浓度为4μmol／L，作用时间为24h；空白对照组：未加任何药物，作用24h。（1）采用逆转录（RT）．PCR技术、蛋白印迹（westernblot）法分别检测3组细胞中GRP78、beclinl、Bcl-2、CHOPmRNA和蛋白的表达；（2）荧光染色法检测3组细胞的自噬情况；（3）流式细胞仪检测3组细胞的凋亡情况；（4）3组细胞经不同浓度（100、50、25、12．5、6．25、3．12、1．56、0．78mg，L）顺铂作用后（即为BAPTA．AM＋顺铂组、A23187＋顺铂组、顺铂组），采用四甲基偶氮唑蓝（MTr）比色法测定3组细胞对顺铂的敏感性[以50％抑制浓度（IC，。）表示]。结果（1）3组SKOV3细胞中GRP78、beclinl、Bc-2、CHOPmRNA和蛋白表达的比较：SKOV3细胞中GRP78、beclinl、Bcl．2、CHOPmRNA表达水平，BAPTA．AM组分别为0．583±0．025、0．860±0．055、0．714±0．032、0．811±0．004，A23187组分另0为0．840±0．044、0．6544-0．065、0．908~0．047、0．620~0．062，空白对照组分别为0．687±0．032、0．772±0．029、0．845±0．018、0．712±0．077；SKOV3细胞中GRP78、beclinl、Bcl-2、CHOP蛋白表达水平，BAPTA—AM组分别为0．423±0．035、0．952±0．022、0．385±0．032、0．681±0．095，A23187组分另0为0．743±0．032、0．638±0．025、0．596±0．029、0．431±0．095，空白对照组分别为0．617±0．031、0．789±0．083、0．492±0．036、0．531±0．003。SKOV3细胞中beclinl、CHOPmRNA和蛋白表达水平，BAPTA．AM组明显高于空白
Objective To explore the effect and mechanism of glucose regulated protein 78 （GRP78） on autophagy and apoptosis in ovarian carcinoma, and to investigate the influence on the growth and sensitivity to eisplatin on the ovarian cancer cells. Methods The human ovarian cancer cell line SKOV3 were treated by the GRP78 regulator BAPTA-AM and A23187, which were used to decrease or increase the expression levels of GRP78, respectively. The experiment were divided into three groups. Cells in the group of BAPTA-AM were treated by BAPTA-AM at the final concerntration of 40μmol/L for 1 hour. Cells in the group of A23187 were treated by A23187 at the final concerntration of 4μmol/L for 24 hours. While, cells in the control group were treated by culture medium without any GRP78 regulator for 24 hours. The expressions of GRP78, beclinl, Bcl-2 and CHOP mRNA and protein were detected by reverse transcription （RT）-PCR and western blot. The autophagy levels was observed by t^een fluorescent protein-microtubule-associated protein 1 light chain 3-Ⅱ（GFP-LC3-Ⅱ ） fluorescence staining. The flow eytometry was used to analyse the apoptosis rates of cells. The effect on cell growth and the sensitivity to eisplatin of SKOV3 were accessed by methyl thiazolyl tetrazolium （MTT）. Results （1）The mRNA expressions of GRP78, beclinl, Bcl-2 and CHOP in the group of BAPTA-AM were 0.583-±0.025, 0.860-± 0.055, 0.714±0.032 and 0.811 ±0.004, respectively. The mRNA expressions of GRP78, beclinl, Bcl-2 and CHOP in the group of A23187 were 0.840 -± 0.044, 0.654 ± 0.065, 0.908 -± 0.047 and 0.620 ± 0.062, respectively. The mRNA expressions of GRP78, beclinl, Bcl-2 and CHOP in the control group were 0.687± 0.032, 0.772-± 0.029, 0.845 ±0.018, 0.712 ±0.077, respectively. While the protein expressions of GRP78, beclinl, Bel-2 and CHOP in the group of BAPTA-AM were 0.423±0.035, 0.952±0.022, 0.385±0.032, 0.681± 0.095, respectively. The protein expressions of GRP78, beelinl, Bcl-2 and CHOP in the group of A23187 were 0.743 �
出处
《中华妇产科杂志》
CAS
CSCD
北大核心
2015年第11期848-853,共6页
Chinese Journal of Obstetrics and Gynecology
关键词
卵巢肿瘤
热休克蛋白质类
自噬
细胞凋亡
顺铂
Ovarian neoplasms
Heat-shock proteins
Autophagy
Apoptosis
Cisplatin
