生存素下調多藥耐藥蛋白增強人鼻咽癌細胞株對紫杉醇敏感性的研究

楊寧+朱樂攀+譚潭+等

楊 寧1 朱樂攀2 譚 潭2 侯春燕1

1.湖南省郴州市第一人民醫院耳鼻喉科，湖南郴州 423000；

2.湖南省郴州市第一人民醫院檢驗科，湖南郴州 423000

[摘要] 目的 研究生存素（Survivin）下調多藥耐藥蛋白（MRP）的表達與鼻咽癌紫杉醇（PTX）耐藥性關系，探討鼻咽癌PTX耐藥的分子機制。 方法 采用免疫組化檢測Survivin和MRP在42例鼻咽癌PTX耐藥患者與24例PTX非耐藥患者中的表達；采用濃度遞增法持續誘導建立鼻咽癌化療耐藥細胞株5-8F-PTX（+），繪制細胞生長曲線，測定細胞生長的倍增時間，檢測腫瘤細胞的周期分布；采用siRNA技術干擾5-8F-PTX（+）中Survivin的表達后，Western blot法檢測Survivin和MRP表達變化，MTT檢測不同抗腫瘤藥物PTX、順鉑（cDDP）、5-氟尿嘧啶（5-FU）、長春新堿（VCR）耐藥敏感性的變化。 結果 Survivin在鼻咽癌化療耐藥患者中陽性表達率為83.3%，明顯高于非耐藥患者（41.7%），差異有高度統計學意義（P < 0.01）；MRP在鼻咽癌PTX耐藥患者中表達陽性率為88.1%，明顯高于非耐藥患者（37.5%），差異有高度統計學意義（P < 0.01）。5-8F-PTX（+）較5-8F的細胞生長速度明顯減慢，5-8F-PTX（+）細胞生長的倍增時間為21 h，親本5-8F細胞生長的倍增時間為15 h；5-8F-PTX（+）的G2/M期細胞百分比[（23.1±1.3）%]顯著高于親本5-8F細胞[（13.5±0.9）%]；Survivin和MRP在PTX耐藥細胞株5-8F-PTX（+）細胞株中的表達水平明顯高于非耐藥的5-8F，siRNA干擾5-8F-PTX（+）中Survivin的表達后，Survivin和MRP表達明顯下調，PTX、cDDP、5-FU、VCR耐在siRNA-5-8F-PTX（+）中的IC50值不同程度地下降。 結論 Survivin可通過下調MRP的表達增強鼻咽癌細胞對PTX的敏感性。

[關鍵詞] 鼻咽癌；紫杉醇；耐藥性；生存素；多藥耐藥相關蛋白

[中圖分類號] R739 [文獻標識碼] A [文章編號] 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.

[Key words] NPC； Paclitaxel； Drug resistance； Survivin； MRP

鼻咽癌（NPC）是頭頸部腫瘤中常見的惡性腫瘤，在中國南方其發病率居世界首位[1]。臨床上，鼻咽癌確診時往往已經到了晚期，其臨床治療方式是放療和化療相結合的方法，其臨床療效比較差，其5年無病生存率小于60%，化療耐藥的產生是鼻咽癌化療失敗的主要原因之一[2]。……