新型纳米基因载体的制备及其在乳腺癌治疗中的应用研究

Gene therapy has provided a broad space for the treatment of hereditary diseases, disorders of the immune system and tumors.The bottleneck of the clinical application of gene therapy is to find a secure and effective way to import, and at present a great deal of work is dedicated to bioactive marcomolecules and nano-materials.They can combine with DNA molecules to form complexes, via which target genes can be transformed into target cells safely and effectively, achieving the purposes of tissue repair or gene therapy. The current research about transfection mediated by nano-materials mainly focuses on degradable biological macromolecules, silicon dioxide, cationic macromolecules and so on, while little on calcium carbonate materials. Our work is based on a new calcium carbonate Nanoparticle（ACC/CaIP6）,which has been patented by China(Patent NO. 200910193766.5). With high specific surface area, the ACC/CaIP6 Nanoparticle can bind DNA and transfecte cell effectively,and has no negative impact on cells. Use such material as substrate, which modified with cationic material, to make the surface with appropriate positive charges,which can occur a better interaction with DNA and improve the transfection efficiency . Moreover, we combine breast cell specific expression HSV-tK suicide gene system with ACC/CaIP6 Nanoparticle，through both in vitro and in vivo experiments, to investigate the appllication of ACC/CaIP6 Nanoparticle in breast cancer gene therapy, to explore its potential clinical application.

近年来，基因治疗在乳腺癌治疗中显示出良好的应用前景，但由于缺乏安全高效的基因载体，其临床应用受到很大限制。纳米载体具有生物兼容性、可生物降解、药物缓释和药物靶向等优点，已成为基因治疗研究的热点。申请人多年来致力于研究和开发新型纳米基因载体，前期研究合成了具有高比表面积的无定型碳酸钙纳米材料（ACC/CaIP6），并获得国家专利（200910193766.5）。该纳米材料表面带正电荷，可高效结合DNA并在体外成功转染乳腺癌细胞，对细胞基本无毒性。我们拟根据现有成果加入阳离子表面活性剂对ACC/CaIP6纳米材料进行表面修饰，力求制备出具更高DNA结合率及细胞转染率的新型纳米材料；进一步结合HSV-tK自杀基因质粒制备ACC/CaIP6/HSV-tK复合物，在乳腺癌细胞及动物模型中观察其对肿瘤的杀伤作用，为ACC/CaIP6纳米载体应用于乳腺癌及相关肿瘤的治疗提供新的思路和实验依据。

为研究新型纳米载体在乳腺癌治疗中的作用,本研究运用前期研究合成了具有高表面积的无定型碳酸钙纳米材料（ACC/CaIP6），进行体外，体内试验。首先通过电子扫描显微镜，确定制备的纳米颗粒ACC/CaIP6的特性。琼脂糖凝胶电泳试验，确定纳米颗粒ACC/CaIP6与siRNA结合的最佳质量比为50:1。MTT分析，ACC/CaIP6和siRNA组成的复合物对MCF-7细胞毒性低。用流式细胞术分析ACC/CaIP6/FAM-siRNA在质量比为50:1 150 nM FAM-siRNA和Lipofectamine 2000/FAM-siRNA的转染效率近似一致。在MCF-7细胞中，通过转染ACC/CaIP6/siAKT1，MTT分析表明敲低AKT1后MCF-7细胞的增殖受到抑制。体内试验结果表明注射ACC/CaIP6/siAKT1可抑制肿瘤生长。综合以上结果，本研究为ACC/CaIP6纳米载体应用于乳腺癌及相关肿瘤的治疗提供新的思路和试验依据。项目资助发表论文11篇，发明专利4项，项目投入经费70万元，支出62.1423万元，各项支出基本与预算相符。剩余经费7.8577万元，计划用于本项目研究后续支出。