慢病毒介导九香虫Cyt-c基因对乳腺癌细胞体内外生长抑制作用研究

Breast cancer seriously harms human health. Aspongopus chinensis Dallas has been used as a traditional Chinese medicine and exhibits good clinical anti-tumour effects. After eight years of systematic research, Our research team has purified and obtained an anti-tumor protein named cytochrome C (Cyt-c) for the first time, and made its anti-tumor mechanism to be clear, which shows good prospects for application in anti-cancer. However, Cyt-c is water-soluble with a large molecular weight and hardly penetrate through the cell membrane. Therefore, how to improve the bioavailability of Cyt-c during administration is a key problem that needs to be solved. This project intends to use the emerging gene therapy technology to carry out the in-depth development and utilization of Cyt-c from the traditional Chinese medicine A. chinensis Dallas. According to the characteristics of the Lentivirus HIV-1 Gag that can self-assemble and carry other macromolecules into the virus particle, We can cloned gene of Cyt-c from A. chinensis Dallas, then Cyt-c gene is carried by lentiviral vector, When the virus is packaged and infected with breast cancer cells, Cyt-c will also be packaged with Gag into viral particles and enter into breast cancer cells for expression, so as to achieve the purpose of treatment of breast cancer. In addition, this project will also investigate the anti-cancer effects of Cyt-c on transplanted tumors of breast cancer in mice, so as to provide experimental basis for developing safe, highly effective anti-tumor drugs with independent intellectual property rights, and provide reference for Development and utilization of other active peptide from traditional Chinese medicine at the same time.

乳腺癌严重危害人类健康，传统中药九香虫具有确切的抗肿瘤应用基础，本研究团队经过八年的系统研究，首次从九香虫中纯化获得一种抗肿瘤蛋白—细胞色素C（Cyt-c），并明确了其抗肿瘤机理，显示出良好的应用前景。但Cyt-c为水溶性，分子量较大，不易透过细胞膜，如何提高Cyt-c在给药过程中的生物利用度是亟需解决的关键问题。本项目拟采用新兴的基因治疗技术对传统中药九香虫Cyt-c进行深层次开发利用，根据慢病毒HIV-1 Gag可自我组装并运载其他大分子到病毒颗粒中的特性，通过克隆九香虫Cyt-c基因，利用慢病毒载体携带Cyt-c，在病毒包装及感染乳腺癌细胞时，Cyt-c随Gag包装入病毒颗粒并导入乳腺癌细胞中表达，从而达到治疗乳腺癌的目的。此外，本项目还将探讨Cyt-c对乳腺癌小鼠移植瘤的抗癌效果，为开发安全、高效、拥有自主知识产权的抗肿瘤药物提供实验基础，同时为其他传统中药的开发利用提供参考。

乳腺癌严重危害人类健康，传统中药九香虫具有确切的抗肿瘤应用基础，本研究从九香虫中纯化获得Cytochrome c（Cyt c），可抑制乳腺癌细胞的体内外增殖，显示出良好的应用前景。但Cyt c为水溶性，分子量较大，不易透过细胞膜，如何提高Cyt c在给药过程中的生物利用度是亟需解决的关键问题。本项目根据慢病毒HIV-1 Gag可自我组装并运载其他大分子到病毒样颗粒中的特性，通过克隆九香虫Cyt c基因，利用慢病毒载体携带Cyt c，在病毒样颗粒包装及感染乳腺癌细胞时，Cyt c随Gag包装入病毒样颗粒并导入乳腺癌细胞中表达，从而达到治疗乳腺癌的目的，为开发安全、高效、拥有自主知识产权的抗肿瘤药物提供实验基础，同时为其他传统中药的开发利用提供参考，取得的主要研究结果如下：.1、九香虫Cyt c蛋白对乳腺癌细胞体内外生长的影响.低剂量Cyt c（5 µg/mL）可显著促进乳腺癌细胞增殖（P<0.05）；中、高剂量Cyt c（10 µg/mL与20 µg/mL）可显著抑制乳腺癌细胞体外增殖（P<0.05），九香虫Cyt c（20 mg/kg/2 d）可抑制乳腺癌移植瘤的体内生长。.2、携带九香虫Cyt c慢病毒表达质粒的构建与病毒样颗粒包装.构建了携带九香虫Cyt c的慢病毒表达质粒pWSLV-Cyt c，制备了病毒样颗粒，荧光显微镜下可见红色荧光蛋白mcherry的表达；Western blot可检测到病毒衣壳蛋白p24的表达。.3、携带九香虫Cyt c的病毒样颗粒对乳腺癌细胞体外生长的影响.在病毒样颗粒mcherry-Cyt c-VLP处理组中CFDA-SE绿色荧光的细胞数量较少，荧光较强，表明mcherry-Cyt c-VLP抑制了乳腺癌细胞增殖。流式细胞术与透射电镜检测结果显示，mcherry-Cyt c-VLP可诱导MDA-MB-453细胞凋亡。.4、携带九香虫Cyt c的病毒样颗粒对乳腺癌细胞体内生长的影响.病毒样颗粒mcherry-Cyt c-VLP可显著降低乳腺癌移植瘤小鼠瘤体的体积与重量，诱导乳腺癌移植瘤细胞凋亡，脾、肺、肾HE染色组织切片镜检未见异常。.综上，低剂量九香虫Cyt c可促进乳腺癌细胞生长，中、高剂量九香虫Cyt c可抑制乳腺癌细胞的生长；慢病毒介导九香虫Cyt c可显著抑制乳腺癌细胞体内、外生长，并可诱导乳腺癌细胞凋亡，提高了抗肿瘤效果。

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