基于靶向和药物共载的Bmi-1RNAi壳聚糖-聚乙二醇纳米微粒的构建及其对喉癌实体瘤干细胞杀伤机制研究

A growing body of evidence has been reported that a tumor clone is organized as a hierarchy that originates from rare stem cells in solid laryngeal cancer stem cell. Those stem cells have the capacity of self-renewal and multilineal differentiation potency.Its resistant to traditional therapy is the reason of cancer recurrence and metastatic. Curative therapies are possible only when these cells are targeted. The recent research have comfirmed proto-oncogene Bmi-1 is key gene of self-renewal and multilineal differentiation in laryngeal cancer stem cells. Silencing Bmi-1 can enhance the sensitivity of CD133+ tumor stem cells to chemotherapy. In this study, the recombinant plasmid vector Bmi-1RNAi and RGD peptide-modified Chitosan-Polyethyleneglycol palliate-loaded nanoparticles will be constructed. Then the nanoparticles will be transfected into laryngeal cancer stem cells. The differentiation,cell proliferation and tumor-forming ability of cancer stem cells will be observed, the changes of the signal pathway and cell cycle will be monitored. The mechanism of cRGD peptide，paclitaxel and Bmi-1RNAi gene multiply targeted killing the laryngeal cancer stem cells will be investigated.This research will verify targeted knockout the Bmi-1 gene,combined with cRGD peptide，paclitaxel can downregulate Bmi-1 pathway and kill the laryngeal cancer stem cells multiply,so make it possible to eliminate laryngeal cancer. This research will be the way to discover new nanoparticles drugs for treating laryngeal cancer.

研究已证实喉癌组织中存在少量具多分化潜能的肿瘤干细胞，它们对传统治疗的耐受是喉癌复发、转移的主要原因；靶向杀伤喉癌干细胞已是喉癌治疗的共识。申请者的前期研究及文献证实，原癌基因Bmi-1是喉癌干细胞增殖与分化调控的关键位点，用Bmi-1RNAi方法敲除Bmi-1基因，可抑制喉癌干细胞，使其对化疗药物的敏感性增强。但单纯Bmi-1-RNAi核苷酸体内易降解，缺乏靶向性，是限制该技术临床转化的瓶颈。本项目拟在前期研究的基础上，构建制备cRGD肽修饰的共载紫杉醇与Bmi-1RNAi质粒的壳聚糖-聚乙二醇纳米微粒，转染喉癌干细胞，以期证实该纳米微粒可双重靶向喉癌干细胞，下调Bmi-1基因，联合紫杉醇化疗精准杀伤喉癌干细胞；监测喉癌干细胞“干性”改变及相关信号通路，探讨cRGD肽、紫杉醇与Bmi-1RNAi多重靶向杀伤喉癌干细胞的重叠增效机制，为喉癌干细胞生物治疗的临床转化奠定基础。

研究已证实喉癌组织中存在少量具多分化潜能的肿瘤干细胞，它们对传统治疗的耐受是喉癌复发、转移的主要原因；靶向杀伤喉癌干细胞已是喉癌治疗的共识。课题组前期研究及文献证实，原癌基因Bmi-1是喉癌干细胞增殖与分化调控的关键位点，用Bmi-1RNAi方法敲除Bmi-1基因，可抑制喉癌干细胞，使其对化疗药物的敏感性增强。但单纯Bmi-1-RNAi核苷酸体内易降解，缺乏靶向性，是限制该技术临床转化的瓶颈。本项目在前期研究的基础上，制备cRGD肽修饰的共载紫杉醇与Bmi-1RNAi基因的壳聚糖-聚乙二醇缓释纳米微粒 (cRGD/CS-PEG-Bmi-1RNAi-PTX)，微粒近似球形，分布均匀，粒径200±10.20 nm大小，有良好的的载体DNA包裹能力和抗核酸酶降解能力，包封率（85.01 ± 1.64）%，载药量为（4.21 ± 0.63）%，基因沉默效率约为75±4.23%；mPEG-CS-cRGD/Bmi-1RNAi-PTX纳米微粒在体外对喉癌 Hep-2 细胞的迁移、增殖及集落形成能力均有良好的抑制作用，且可以促使其细胞凋亡，纳米微粒作用后的Hep-2 细胞，CD133+干细胞细胞比例降低；本研究亦使用化疗药物紫杉醇筛选富集Hep-2喉癌干细胞，富集后的Hep-2细胞，集落形成能力及干细胞相关基因表达上调，具干细胞特点；纳米微粒对富集干细胞干性基因表达有明显的抑制作用，并使凋亡基因升高，抗凋亡基因、肿瘤黏附浸润相关基因下调，具有明显的肿瘤抑制作用；建立人喉癌动物模型，小鼠尾静脉注射纳米微粒悬液，与对照组比较，对小鼠喉癌移植瘤有明显的抑制作用，且治疗组体重稳定，生命重要器官心、肾、肝均未见明显异常，仅瘤体组织出现大片状坏死；检测知治疗组瘤体中干细胞相关蛋白，肿瘤浸润及凋亡相关蛋白均表达下调，可见，cRGD肽、紫杉醇与Bmi-1RNAi基因可以多重靶向杀伤喉癌干细胞，本研究将为喉癌干细胞生物治疗的临床转化提供实验基础。