聚酰胺-金纳米偶联物的构建及其下调PD-1基因诱导T细胞功能恢复和c-Myc基因促进癌细胞凋亡的机制

Polyamides, mainly composed of 1-methylpyrrole and 1-methylimizole, can specifically recognize predetermined DNA sequences in the minor groove and regulate the expression of genes. To solve the key questions on polyamide drugability: poor permeability of polyamides, specificity of recognizing longer gene sequences and in vitro & in vivo detailed action mechanism of polyamides with targeted genes, The project is involved with: (1) constructing 14 chirally-modified polyamide-gold nanoparticle conjugates with structural novelty; (2) at an extracellular level examining the regulation ability and characteristics of conjugates specificially binding PD-1 gene overexpressed in HIV-infected T cells and c-Myc gene overexpressed in three kinds of cancer cells, human small cell lung carcinoma, breast cancer, chronic myeloid leukemia, and at an intracellular level the ability of target downregulating PD-1 gene inducing recovery of T cell immune function and c-Myc gene inhibiting proliferation of and promoting apoptosis of cancer cells by integrated interdisciplinary techniques, such as SPR, Molecular Simulation (MSi), Gel electrophoresis, CD techniques and melting Tm analysis, RT-PCR, Western Blotting, Real-time Cell Analysis, Would Healing and so on, and clarifying their interaction molecular mechanism. More importantly, employing the conjugates for coupling polyamides to surface of gold nanoparticles is attempting to increase the permeability of polyamides and the whole regulation ability to targeted genes. Through conducting the project, it is of importance for seeking for small molecular targeted drugs to regulate PD-1 and c-Myc genes, in particular, providing a novel idea for polyamides’ study via a combination of gold nanoparticles with polyamides. Aims for the project are: to understand the mechanism and setting up a technique for synthesizing conjugates; to explore drugability of chirally-modified conjugates targeted to PD-1 and c-Myc genes and regulation mechanism; and to search for the functionalization and interaction nature of AuP in the polyamide-DNA sequence recognition event.

聚酰胺特异性地识别Ｂ-DNA序列及调控基因的表达。针对聚酰胺成药性中面临的关键问题：透膜性差、识别长链基因序列的特异性及体内外调控机制。本项目：①构建14个聚酰胺-金纳米颗粒（AuP）偶联物；②综合多学科交叉技术，考察偶联物在细胞外对PD-1基因、c-Myc基因DNA的特异性键合能力和特征；以及在细胞内靶向下调在HIV感染的T细胞中高表达的PD-1基因、诱导T细胞免疫功能恢复，及在三种癌细胞中高表达的c-Myc基因、抑制细胞增殖、促进细胞凋亡的能力，阐明分子机制。重要的是：通过金纳米粒子的引入，试图增加聚酰胺的透膜性，提高靶基因的调控能力及探索聚酰胺研究新思路。项目的实施，对加速聚酰胺靶向基因的治疗药物研发意义重大。目标：探明制备偶联物的反应机制，建立合成它们的技术方法；探求偶联物靶向PD-1和c-Myc基因的成药潜力，以及调控能力和机制；探寻AuP在聚酰胺-DNA识别事件中的作用规律。

聚酰胺能特异性地识别Ｂ-DNA序列及调控基因的表达。并且，具有相对良好的细胞渗透性、能进入核中，不易被核酸酶水解，相对转录因子能较强的键合特定靶基因。充分的实验数据证明: 它能通过在启动子区和增强子上干涉转录系统，达到调控与疾病相关突变基因的表达，而对正常细胞在实验条件下没有损伤。针对聚酰胺成药性中面临的关键问题：透膜性差、识别长链基因序列的特异性及体内外调控机制。本项目：（1）首先探索合成克级级以上规模的聚酰胺制备工艺，为进一步合成聚酰胺-金纳米颗粒（AuP）偶联物提供充足的原材料和对比化合物；（2）综合多学科交叉技术，考察聚酰胺及偶联物在细胞外对PD-1基因、c-Myc基因DNA的特异性键合能力和特征；（3）在细胞内考察偶联物靶向调控在HIV感染的T细胞中高表达的PD-1基因、诱导T细胞免疫功能恢复，及考察在小细胞肺癌、骨髓白血病、乳腺癌三种肿瘤细胞中高表达的c-Myc基因、抑制细胞增殖、促进细胞凋亡的能力，阐明分子机制。通过金纳米粒子的引入，试图增加聚酰胺的透膜性，提高靶基因的调控能力及探索聚酰胺研究新思路。除此之外，项目组在项目实施中由于考虑PD-1和c-Myc两种基因启动子区有段富含鸟嘌呤的DNA序列，在一定条件下，有形成G-四链体，从而调控基因表达，抑制细胞增殖的潜力。所以，基于项目组前期研究基础，我们设想能否通过其他比聚酰胺偶联物分子小的分子，用于诱导G-四链体形成，达到调控基因表达的目的。因此，项目组考察了菲罗琳和4，5-二氮芴类衍生物的抗肿瘤及靶向G-四链体的能力，并取得了较好的初步实验结果，目前，正应用这些分子的本项目后续研究中。由于纯化偶联物遇到困难，透膜性不能很好解决，致使项目进展不顺利和延迟实验结果产出。本项目细胞内药物活性及作用机制还有待进一步考察。项目的实施，初步摸索出合成偶联物的规律及胞外靶向PD-1/c-Myc基因的作用性质和能力。这些工作对加速聚酰胺靶向基因的治疗药物研发意义重大。

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