杨 宁1 朱乐攀2 谭 潭2 侯春燕1
1.湖南省郴州市第一人民医院耳鼻喉科，湖南郴州 423000；
2.湖南省郴州市第一人民医院检验科，湖南郴州 423000
[摘要] 目的 研究生存素（Survivin）下调多药耐药蛋白（MRP）的表达与鼻咽癌紫杉醇（PTX）耐药性关系，探讨鼻咽癌PTX耐药的分子机制。 方法 采用免疫组化检测Survivin和MRP在42例鼻咽癌PTX耐药患者与24例PTX非耐药患者中的表达；采用浓度递增法持续诱导建立鼻咽癌化疗耐药细胞株5-8F-PTX（+），绘制细胞生长曲线，测定细胞生长的倍增时间，检测肿瘤细胞的周期分布；采用siRNA技术干扰5-8F-PTX（+）中Survivin的表达后，Western blot法检测Survivin和MRP表达变化，MTT检测不同抗肿瘤药物PTX、顺铂（cDDP）、5-氟尿嘧啶（5-FU）、长春新碱（VCR）耐药敏感性的变化。 结果 Survivin在鼻咽癌化疗耐药患者中阳性表达率为83.3%，明显高于非耐药患者（41.7%），差异有高度统计学意义（P < 0.01）；MRP在鼻咽癌PTX耐药患者中表达阳性率为88.1%，明显高于非耐药患者（37.5%），差异有高度统计学意义（P < 0.01）。5-8F-PTX（+）较5-8F的细胞生长速度明显减慢，5-8F-PTX（+）细胞生长的倍增时间为21 h，亲本5-8F细胞生长的倍增时间为15 h；5-8F-PTX（+）的G2/M期细胞百分比[（23.1±1.3）%]显著高于亲本5-8F细胞[（13.5±0.9）%]；Survivin和MRP在PTX耐药细胞株5-8F-PTX（+）细胞株中的表达水平明显高于非耐药的5-8F，siRNA干扰5-8F-PTX（+）中Survivin的表达后，Survivin和MRP表达明显下调，PTX、cDDP、5-FU、VCR耐在siRNA-5-8F-PTX（+）中的IC50值不同程度地下降。 结论 Survivin可通过下调MRP的表达增强鼻咽癌细胞对PTX的敏感性。
[关键词] 鼻咽癌；紫杉醇；耐药性；生存素；多药耐药相关蛋白
[中图分类号] R739 [文献标识码] A [文章编号] 1673-7210（2014）03（b）-0009-06
The study of Survivin increase drug sensitivity to Paclitaxel in human nasopharyngeal carcinoma cell line by down regulate MRP expression
YANG Ning1 ZHU Lepan2 TAN Tan2 HOU Chunyan1
1.Department of ENT， the First People"s Hospital of Chenzhou City， Hu"nan Province， Chenzhou 423000， China； 2.Department of Inspection， the First People"s Hospital of Chenzhou City， Hu"nan Province， Chenzhou 423000， China
[Abstract] Objective To find the relationship among Survivin and multidrug resistance associated protein （MRP） and drug resistance in nasopharyngeal carcinoma （NPC）， and to explore the mechanism of drug resitance to Paclitaxel （PTX） in NPC. Methods Expression of Survivin and MRP were detected by immunohistochemistry method in 42 cases of NPC patients with Paclitaxel resistance and 24 cases of NPC patients without Paclitaxel resistance. The Paclitaxel resistance cell line， 5-8F-PTX （+） was established by a step-increased method. The curve of growth were drew and the doubling time were detected， and the distribution of cell cycle were detected by flow cytometry in 5-8F-PTX （+） and 5-8F. The expression of Survivin and MRP were detected by western blot after siRNA to 5-8F-PTX （+）， the drug sensitivity of various kinds of antitumor drug， such as Paclitaxel， cDDP， 5-FU and Vincristine were detected by MTT method. Results The positive of survivin were 83.3% in NPC patients with Paclitaxel resistance and significant high than that of NPC patients without Paclitaxel resistance （41.7%）， the difference was highly statistically significant （P < 0.01）； the positive of MRP was 88.1% in NPC patients with Paclitaxel resistance and significant high than that of NPC patients without Paclitaxel resistance （37.5%）， the difference was highly statistically significant （P < 0.01）. The growth velocity decreased more obviously in 5-8F-PTX （+） than 5-8F， and the doubling time were 21 h in 5-8F-PTX （+） and 15 h in parent 5-8F. The ratio of G2/M cell cycle [（23.1±1.3）%] in 5-8F-PTX （+） was higher than that in 5-8F [（13.5±0.9）%]. The expression of Survivin and MRP were higher in 5-8F-PTX （+） than those in 5-8F. After siRNA treatment， expression of Survivin and MRP were obviously down regulated in siRNA-5-8F-PTX （+） than 5-8F-PTX （+） and 5-8F-PTX （+）-empty vector. The IC50 of Paclitaxel， cDDP， 5-FU and Vincristine were significantly down regulated in siRNA-5-8F-PTX （+） than 5-8F-PTX （+）. Conclusion Decreasing of survivin expression can down regulate the expression of MRP and result into increase of drug sensitivity to Paclitaxel in NPC.