低能量超声联合微泡调节自噬对耐紫杉醇卵巢癌作用及机制研究

Paclitaxel-resistance remains one of the key reasons for treatment failure and death associated with ovarian cancer. Exploring the mechanisms of drug resistance and reversing drug resistance is a today’s hot topic. The several studies have shown that low intensity ultrasound with microbubble (UTMD) combined with conventional chemotherapeutic drugs can effectively reverse the drug resistance, but the mechanism has not yet been clarified. Our previous studies showed the relationship between paclitaxel-resistant ovarian cancer cell and autophagy regulation. We also showed UTMD significantly enhanced the chemotherapeutic effect on paclitaxel-resistant ovarian cancer cells leading to the decrease of the cell survival rate, and upgraded the autophagic level. Interestingly, the effect was been weakened by the inhibitor of autophagy. This phenomenon was illustrated that UTMD reversed drug resistance and promoted cell death via the activation of autophagy. In this study, we will apply various methods, such as western blot, confocal fluorescence microscope and transmission electron microscope, to confirm the phenomenon of UTMD-induced autophagy. Moreover, we will screen and select out the UTMD-induced autophagy pathways and key regulatory proteins through the autophagy-related PCR chip. We will apply the shRNA to regulate the expression of key proteins and observe the subsequent effect of UTMD-induced autophagic level on drug-resistant ovarian cancer cell and the paclitaxel sensitivity. Furthermore, the therapeutic effect and safety of UTMD-induced autophagy will be verified in animal model, for the purpose of providing a scientific basis for new treatment strategies of paclitaxel-resistant ovarian cancer.

紫杉醇耐药是卵巢癌治疗失败和死亡的最主要原因之一，探寻耐药机制及逆转耐药策略是目前研究的热点。研究显示低能量超声联合微泡（UTMD）与常规化疗药物联合可有效逆转肿瘤耐药，但目前机制尚未阐明。我们前期研究发现卵巢癌紫杉醇耐药与自噬相关，且UTMD可显著增加耐药细胞对紫杉醇的敏感性，降低细胞存活率，过度激活细胞自噬水平，而当抑制自噬后，UTMD的化疗增敏作用被削弱，提示UTMD通过激活自噬促进细胞死亡，逆转化疗耐药。本项目拟采用Western Blot、共聚焦荧光显微镜、透射电镜等技术检测UTMD所致的细胞自噬水平改变；通过自噬相关PCR芯片筛选UTMD调控自噬的通路和关键调控蛋白，用shRNA调控关键蛋白表达并观察对UTMD所致的细胞自噬水平和紫杉醇敏感性的影响；最后动物模型在体验证治疗的有效性和安全性。旨在揭示UTMD对逆转卵巢癌耐药的潜在机制，为以调节自噬为目的的超声治疗提供科学依据。

紫杉醇耐药是卵巢癌治疗失败和死亡的最主要原因之一，探寻耐药机制及逆转耐药策略是目前研究的热点。本研究在构建的耐紫杉醇卵巢癌细胞株SKOV3-TR和A2780-TR的基础上，首先证明了耐紫杉醇卵巢癌细胞的高自噬表达水平，并通过Western Blot、共聚焦荧光显微镜、透射电镜等技术展示了低强度聚焦超声联合微泡(LIFU+MB)对耐紫杉醇卵巢癌细胞自噬水平的下调，以及从而增加了对紫杉醇药物的敏感性，体现在细胞实验和动物实验的肿瘤生长受到抑制。进而使用RNA-seq筛选LIFU+MB调控自噬的相关通路，进一步通过qRT-PCR验证，筛选出LIFU调控的P13K/AKT/mTOR等自噬相关通路，mTOR可能是关键调控分子。基于结果，会议报告2次，完成论文直接相关期刊论文撰写2篇，其中一篇处于审稿状态。在项目执行中，不断优化超声研究实验设备，自主研发水槽式和手持式低强度聚焦超声发生仪，授权国家实用新型专利2项，申请国家实用新型专利2项。项目申请人和参与人二人被列入省级人才培养资助。

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