纳米抗体靶向型载体介导miR122诱导非小细胞肺癌凋亡的分子机制研究

Specific targeted therapy for cancer is a hot field in medical research. In the project, nanobody with high specificity, low molecular weight and immunogenicity was employed, and high efficient and safe non-viral gene carrier was constructed via the modification of PEI25K by a hydrophobic amino acid N-acetyl-L-leucine and chemically conjugation via NHS-PEG5K-Mal method to a nanobody targeting CEACM6 molecules expressed in a higher level on the surface of non-small cell lung cancer cells. The carrier was of high gene transfection efficiency and low toxicity, and could realize the miR-122 delivery. The system was of high stability and active target, and could block the antigen and interference the expression of Bcl-2 to inhibit the proliferation and metastasis of non-small cell lung cancer. In conclusion, the project established a novel route for preparing efficient and stable gene carriers for understanding more then two ways to kill cancer cells, and would facilitate the development and application of non-viral gene carriers and their clinic application in cancer gene therapy.

针对肿瘤的特异性靶向治疗是当今肿瘤医学研究领域的热点，而利用纳米抗体的特异性高、分子量小、免疫原性低等特点，以及新型高效、安全的非病毒基因传输载体所构建的诱导非小细胞肺癌凋亡的靶向传输载体是本研究的核心内容。本项目首先通过疏水性氨基酸N-乙酰-L-亮氨酸对PEI25K的修饰及NHS-PEG5K-Mal为桥梁的化学键合策略，将针对非小细胞肿瘤细胞表面高表达CEACAM6的纳米抗体与之偶联，同时负载具有抑癌作用的miR-122构建成靶向型基因传输载体；该载体具有稳定、主动靶向以及通过封闭肿瘤细胞高表达抗原活性和干扰胞内Bcl-2表达的双重作用效果来抑制非小细胞肺癌的增殖和转移、诱导肿瘤细胞凋亡。本项目的实施，将构建一条高效、稳定的肿瘤靶向型基因递送体系合成路径，并利用该载体探讨特异性多重杀伤肿瘤的分子机制，为靶向型基因治疗药物的研究开发及肿瘤基因治疗的临床应用提供新的思路。

特异性识别癌胚抗原6的纳米抗体具有分子量小，结构简单的特征。 它以卓越的物理化学性质，人性化的可能性以及独特的抗原识别性质使其成为构建靶向递送载体优良候选物。本研究以人乳腺癌细胞MCF-7为模型，采用单域抗体（Nb）修饰的高分子PEN作为miR-34a的递送载体，系统评价对乳腺癌细胞增殖和迁移的抑制作用。 在成功递送miR-34a之后，由于细胞凋亡和细胞周期阻滞的激活，细胞增殖受到明显抑制。 同时，PEN-Nb介导的miR-34a递送可实现通过伤口愈合和transwell迁移测定阐明的对细胞迁移的抑制。 因此，PEN-Nb可能被用作肿瘤靶向系统，用作实现肿瘤基因治疗的miR-34a递送。