携载PDLIM5 siRNA超声纳米微泡靶向调控AMPK/mTOR信号通路拮抗NSCLC耐药研究

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) could inhibit the proliferation and differentiation of tumor cells via blocking the downstream signaling pathway and have been become a first-line treatment for non-small cell lung cancer (NSCLC). However, NSCLC is prone to get acquired resistance to EFGR-TKIs through target gene modification and alternative pathway activation. PDLIM5 could induce tumor cell autophagy, maintain its survival and antagonize EGFR-TKIs, having been a major mechanism of EGFR-TKIs resistance by activating AMPK/mTOR signaling. Ultrasound nanobubbles with targeting specificity could carry gene/drugs and release in control. Combined mechanisms of NSCLC acquired resistance, siRNA technology and our previous research results of ultrasound microbubbles, we intend to design PDLIM5 siRNA-loaded ultrasound nanobubbles to inhibit PDLIM5 expression to control cell autophagy which mediated by AMPK/mTOR activation. And then enhance the sensitivity of NSCLC to EGFR-TKIs, inhibit the growth of EGFR-resistant cell lines or tumor. Therefore, we hope to provide new approaches and means of treatment for the study of drug resistance mechanisms of NSCLC, development of new therapeutic targets and targeted drugs.

表皮生长因子受体酪氨酸激酶抑制剂（EGFR-TKIs）阻断其下游信号传导，抑制肿瘤细胞的异常增殖与分化、抑制肿瘤生长与进展，已成为非小细胞肺癌（NSCLC）一线治疗手段。但NSCLC易通过靶基因修饰、旁路信号传导激活对EGFR-TKIs治疗产生获得性耐药，其中PDLIM5介导AMPK/mTOR活化诱发肿瘤细胞自噬、维持细胞存活、拮抗EGFR-TKIs是产生耐药的主要机制。超声纳米微泡技术可携载基因、药物经超声控释具靶向特异性。结合NSCLC耐药机制、siRNA技术及我们前期超声微泡技术研究结果，设计携载PDLIM5 siRNA超声纳米微泡靶向阻抑PDLIM5进而抑制由AMPK/mTOR活化介导的肿瘤细胞自噬，增强NSCLC对EGFR-TKIs的敏感性，抑制耐药细胞株或肿瘤的生长，提高NSCLC药物治疗效果，为NSCLC耐药机制研究、开发新的治疗靶点及靶向治疗药物提供新的理论依据和治疗方式。

本研究在构建靶向控释的非病毒纳米基因载体、联合基因治疗、超声增强药物递送效率、光热治疗等方面开展系统研究，阐明了PDLIM5基因表达及下游信号通路在非小细胞肺癌（NSCLC）对吉非替尼耐药过程中扮演的重要角色，并有效地抑制了NSCLC细胞对吉非替尼的耐药性。主要包括以下三个方面：1. 超声辐照联合PDLIM5 siRNA-纳米泡靶向拮抗NSCLC细胞耐药：以阳离子脂质（DOPTA）为外壳材料并封装SF-6作为气芯构建新型纳米siRNA递送系统，该体系制备方法简单易行、联合超声可实现靶向控释、具有低毒性和高稳定性等优点，同时超声辐照下因空化效应具有超声增强造影性能；2. 超声介导负载PDLIM5 siRNA的介孔二氧化硅纳米颗粒通过减弱EMT抑制NSCLC细胞吉非替尼耐药：在第一部分所研发的基础上，使用具有高度的生物相容性和易于生物降解、更优的药物负载性能和优良的超声波成像性能的中空介孔二氧化硅作为纳米泡的外壳，构建MSN-siRNA@PVA纳泡体系。中空介孔二氧化硅纳米泡在超声辐照下可以通过界面纳米气泡产生更多的微泡，具有更强的超声对比度。而PDLIM5表达的降低可通过抑制EMT通路恢复NSCLC细胞对吉非替尼的敏感性；3. cRGD靶向金基纳米粒子通过低温光热疗法联合声动力疗法治疗NSCLC的EGFR-TKI耐药：构建了基于球形金纳米颗粒的联合SDT和低温PTT的肿瘤靶向cRGD-GIPG纳米颗粒，用于治疗NSCLC的吉非替尼耐药。cRGD-GIPG具有良好的生物安全性和活性肿瘤细胞靶向性。基于US照射和激光照射后活性氧水平和温度升高的原理，其使TGF-/PDLIM5/SMAD耐药信号通路失活，后激活线粒体凋亡通路，从而最终使吉非替尼治疗敏感并杀死耐药肿瘤细胞。