微泡搭载IDH1R132H-siRNA质粒联合超声靶向破碎技术干扰胶质瘤细胞增殖的新方法研究

Gliomas are the most frequent tumors of the central nervous system, radical surgery is very difficult. Although taking surgery, adjuvant radiotherapy and chemotherapy treatment has obtained favourable result, but the effect is still unsatisfactory. In recent years, the study found that patients with glioma there are high frequency of isocitrate dehydrogenase-1 (IDH1) gene mutation phenomenon, and mutation point fixed. IDH1 mutations are useful genetic alterations for the occurrence, development and prognosis of gliomas. Through comprehensive review, hope find a new target to treat glioma. For the key genes and molecular for targeted molecular therapy has become the research hotspot, but high efficiency and well-targeted gene delivery systems are the key to molecular therapy. As a new type of gene delivery vehicle, ultrasound microbubble contrast agent has unique advantages.This research is IDH1 gene fixed mutation point R132H that closely related with glioma growth as target, combining novel RNA interference technology and ultrasound targeted microbubble destruction technology, made targeted nanoscale microbubble ultrasound contrast agents, from molecular, in vitro cell and animal model in the three level, more comprehensive observation targeted ultrasound microbubble as gene therapy vectors targeted interference glioma cell proliferation, discussion the feasibility of IDH1R132H as a target for glioma gene therapy.

胶质瘤是最常见的颅内肿瘤，手术根治十分困难，虽然采取手术、放化疗等联合治疗取得了一定效果，但疗效仍然不尽如人意。近年研究发现，胶质瘤患者存在高频率的异柠檬酸脱氢酶-1 (IDH1) 基因突变现象，并突变点固定，而且该突变与胶质瘤的发生、发展和预后有明确的关系，对其深入研究，有望找到治疗的新靶点。目前针对关键基因和调控分子为靶点的分子治疗是研究热点，但高效且具有良好靶向性的运载系统是分子治疗的关键，超声微泡造影剂作为一种新型运载工具有独特优势。本研究是以与胶质瘤生长密切相关的IDH1基因固定突变点R132H作为靶点，结合目前新颖的 RNA 干扰技术和超声特有的靶向微泡破碎技术，制备携带基因药物的纳米级靶向微泡超声造影剂，从分子、体外细胞及体内动物模型实验三个水平，较全面观测超声靶向微泡作为基因治疗载体多靶向干扰胶质瘤细胞增殖情况，探讨IDH1R132H作为胶质瘤基因治疗靶点的可行性。

胶质瘤是最常见的颅内肿瘤，手术根治十分困难，虽然采取手术、放化疗等联合治疗取得了一定效果，但疗效仍然不尽如人意。近年研究发现，IDH1基因与胶质瘤的发生、发展和预后有密切的关系，对其深入研究，有望找到治疗的新靶点。目前针对关键基因和调控分子为靶点的分子治疗是研究热点，但如何提高siRNA的转染及干扰效率仍是一大问题。研究表明超声微泡造影剂联合靶向破碎技术，可以有效地提高siRNA的转染和干扰效率。本研究是以IDH1基因为靶点，结合目前新颖的 RNA 干扰技术和超声特有的靶向微泡破碎技术，制备携带基因药物的纳米级靶向微泡超声造影剂，从分子、体外细胞及体内动物模型实验三个水平，较全面观测超声靶向微泡作为基因治疗载体多靶向干扰胶质瘤细胞增殖情况。通过薄膜水化法成功制备出纯的粒径均一的纳米微泡，理化性质良好，符合后期实验要求。激光共聚焦实验显示，纳米微泡联合超声靶向破碎技术能有效提高siRNA的转染效率。PCR及Western-blot结果显示，通过纳米微泡联合超声靶向破碎技术，能有效提高目的基因的干扰效果。流式细胞技术及CCK8细胞活性实验结果显示，实验组（纳米微泡+siRNA）的细胞凋亡明显高于对照（siRNA不加纳米微泡）。体内实验方面，利用荷瘤裸检测纳米微泡对siRNA转染及沉默效率的体内研究，结果显示，实验组（纳米微泡+siRNA）能有效抑制肿瘤生长，延长荷瘤裸鼠的生存期。组织切片结果显示，实验组血管密度明显减低，细胞凋亡明显。综上所述，纳米微泡联合超声靶向破碎技术，能都有效提高siRNA的转染和干扰效率，为siRNA肿瘤治疗提供新思路。