肿瘤响应性核靶向非病毒基因载体的构建及抗肿瘤活性研究

An efficient and safe gene carrier plays a very important role in tumor gene therapy. Cationic non virus gene carrier has the advantages of safety, easy to modify, but problem of hard to through the nuclear membrane resulting in low transfection, lack of tumor response lead to no tumor targeting, restricted the further development of this kind of carrier. In previous studies, a new PAMAM gene carrier linking by disulfide bone has been synthesized , and the carrier of good transfection efficiency has confirmed in vitro transfection experiments. Base on the previous work, this project plan to further introducte a acid sensitive and nuclear targeting molecules aTAT on the SS - PAMAM, the tumor targeting ability of carrier can be further improve by acid sensitive response to the tumor tissue, and by using the TAT cell efficiently penetrate and nuclear targeting effect, the transfection efficiency of carrier can be further improve. We plan to load the antitumor p53 gene, evaluate the targeted ability, anti-tumor effect and safety of the carrier in vivo and in vitro, and study the mechanism of the carrier penetrate membrane and intracellular transport. The design of the gene delivery system and mechanism research has not been reported. This project can provide theoretical basis and experimental instruction for the design of an efficient and safe non virus gene carrier.

一个高效安全的基因载体，是肿瘤基因治疗中一个非常重要的环节。阳离子型非病毒类基因载体具有安全、易于修饰等优点，但其因为难以透过核膜导致转染率低，缺乏肿瘤响应导致肿瘤靶向性差等问题，制约了这类载体的进一步发展。我们在前期的研究中，已经合成得到了通过二硫键连接PAMAM基因载体，通过体外的转染实验已经证实了这一载体良好的转染效率，本课题拟在前期工作的基础上，在SS-PAMAM的上进一步引入酸敏的aTAT核靶向分子，通过酸敏响应进一步提高载体的肿瘤靶向，并利用TAT的细胞高效穿透及核靶向作用，进一步提高载体的转染效果。将其负载具抑癌作用的p53基因，对其靶向性及抗肿瘤效果和安全性进行体内外评价，并研究其穿膜、胞内转运机制。此基因传递系统的设计及作用机制研究国内外尚未见报道。本课题可为高效安全的非病毒基因载体的设计提供理论依据和实验指导。

细胞核是细胞内非常重要的细胞器，是基因编码和转录的场所。然而，针对细胞核靶向的基因转运载体却极少见到报道。因此，设计开发一种高效的核靶向基因载体就显得非常重要。本课题设计合成了一种基于aTAT-SS- PAMAM-D3的基因载体，由于载体表面的aTAT修饰及二硫键的连接，使得载体在进入体内后能够发生尺寸变化和具有核靶向的功能。共聚焦显微镜的观察结果显示经过TAT核靶向分子修饰后，更多的载体进入到了细胞核中，同时二硫键的连接和TAT的修饰使得载体在Hela细胞上达到了更高的转染效率、凋亡率和更低的细胞存活率。然而，只有aTAT-SS- PAMAM-D3载体能够在体内实现长时间的循环和抗肿瘤活性。这一酰胺化TAT和二硫键连接的核靶向载体为我们在肿瘤治疗中高效的基因载体的设计提供了一种新的思路。