【作者】 王颖；

【导师】 曹钟义； 钱永；

【作者基本信息】 南昌大学 ， 口腔医学（专业学位）， 2018， 硕士

【摘要】 目的研究隐丹参酮对舌鳞状细胞癌(TSCC)的抗癌作用并探究其是否可以提高TSCC对紫杉醇的化疗敏感性及其作用机制。方法收集50例舌鳞癌患者的病理组织及临床信息,免疫组织化学染色、评分后分析在舌鳞癌中STAT3表达量的意义。本研究选择两株舌鳞癌细胞系CAL 27、SCC 9进行体外实验。CCK-8实验检测隐丹参酮及紫杉醇单独用药24h和48h的半数有效剂量(EC50),并通过克隆形成实验评估药物对细胞增殖能力的影响。流式细胞术检测药物处理后肿瘤细胞凋亡及周期的改变。使用Transwell、划痕愈合实验检测隐丹参酮、紫杉醇及联合用药处理24h后的细胞侵袭与迁移能力。通过Western印迹测定法检测单独用药及联合用药处理后的细胞内p-STAT3(Ser727)、p-STAT3(Try705)及细胞周期、凋亡及侵袭相关蛋白水平的改变。结果STAT3在肿瘤原发灶中的阳性表达率为44%(22/50),在癌旁组织中则为10%(5/50)。其表达水平与肿瘤临床分期、病理分级及患者是否存在远处转移有密切相关性。Western印迹检测显示,在CAL 27和SCC 9中,STAT3均呈高水平表达。功能学实验(CCK-8,集落形成实验,流式细胞术,transwell实验及划痕愈合实验)结果表明隐丹参酮可以通过抑制STAT3在Try705位点上的磷酸化而抑制舌鳞癌细胞系体外的增殖、侵袭及迁移能力,同时促进细胞凋亡,并使细胞周期阻滞于G2/M期。相比于单独用药组,在联合用药组中表明隐丹参酮可以增强舌鳞癌细胞系对紫杉醇的化疗敏感性,Western印迹显示CDK2、抗凋亡蛋白Bcl-2、转移相关蛋白MMP2表达量均有所下降,而凋亡蛋白Bax,E-cadherin和β-catenin表达上调。结论隐丹参酮可以通过抑制STAT3在Try705位点的磷酸化而降低舌鳞状细胞的恶性增殖、迁移和侵袭能力,同时又能提高舌鳞癌细胞对紫杉醇化疗的敏感性,其可被用作为一种新的治疗策略来克服紫杉醇耐药。更多还原

【Abstract】 Objective In the present study,we further confirmed that STAT3 inhibition is required for the combination treatment with paclitaxel in TSCC cells,suggesting Cryptotanshinone might be a promising candidate for TSCC clinical treatment.Method We examined STAT3 expression levels in neoplastic tissues of TSCC and evaluated the correlation between STAT3 expression and the prognosis of TSCC patients.This study was performed in two tongue squamous cancer cell lines(CAL 27 and SCC 9).The 50% effective dose of Cryptotanshinone and paclitaxel alone was detected by CCK-8 assay and the cell proliferation ability was evaluated using clonogenic assay.Flow cytometry was used to detect cell apoptosis and cycle.The migration and invasion ability were assessed by transwell and scratch assay.The expression levels of p-STAT3(Ser727),p-STAT3(Try705),apoptosis-or cycle-related protein in both cell lines after Cryptotanshinone or combination treatment group were determined by western blot assay.Result The rate of STAT3 expression was 44 %(22/50)in primary foci tissues and 10 %(5/50)in para-cancer tissues,respectively.Furthermore,the expression level of STAT3 is associated with tumor clinical stage,pathologic stage and lymph node metastasis.As a result,western blot assay displayed a high expression of STAT3 in parental cell lines CAL 27 and SCC 9.CCK-8,flow cytometry,transwell migration and scratch assay indicated that Cryptotanshinone attenuated proliferation,invasion and migration capacities and enhanced chemosensitivity to paclitaxel in TSCC cell lines in vitro by inhibiting the phosphorylation of STAT3 Try 705 and Ser 727.Western blot assay showed that down-regulated CDK2,Bcl-2 and Matrix Metalloproteninases(MMPs)protein accompanied by up-regulated Bax,E-cadherin and β-catenin in combination treatment group.Conclusion In conclusion,our findings provide insights that Cryptotanshinone reduces the cell proliferation,invasion and migration ability and sensitizes antitumor effect of paclitaxel on TSCC growth by suppresses phosphorylation of STAT3 Try 705,which might be exploited as a novel therapeutic strategy to suppress paclitaxel resistance.更多还原