磷酸胆碱聚合物MPC_(30)-DEA_(70)负载转化生长因子β1AS-ODN转染心肌细胞

MPC_(30)-DEA_(70)-loaded transforming growth factor beta1 antisense oligonucleotide for transfection of cardiomyocytes

背景:目前反义寡核苷酸的基因治疗已经拥有良好的应用前景,但是反义寡核苷酸的相对分子质量小却不容易入细胞并且易被核酸酶降解,能否有效地穿过细胞膜且不被核酸酶降解从而与目的基因结合发挥作用,因此人们一直致力于理想载体的选择、基因转移效率的增加和转移特异性等研究。目的:探讨新型阳离子磷酸胆碱聚合物MPC30-DEA70能否有效地负载转化生长因子β1反义寡核苷酸(AS-ODN)进入心肌细胞,并观察其对该基因胞内生物表达的影响。方法:按不同N/P电荷比值将载体MPC30-DEA70与转化生长因子β1 AS-ODN络合成形成基因复合物并且对其总电性进行表征;采用MTT法检测MPC30-DEA70与心肌细胞的生物相容性;共聚焦激光扫描显微镜观察MPC30-DEA70/TGF-β1AS-ODN在细胞内的分布和定位;应用流式细胞仪检测MPC30-DEA70/TGF-β1AS-ODN(FAM标记)的细胞转染效率和荧光强度;Western blot、RT-PCR法检测转化生长因子β1细胞内表达水平。结果与结论:MPC30-DEA70与心肌细胞具有较好的细胞相容性,在较高质量浓度(>20 mg/L)下才表现出一定的细胞毒性并呈剂量依赖;MPC30-DEA70/TGF-β1AS-ODN复合物对心肌细胞具有较高的转染效率,并且能够携带转化生长因子β1 AS-ODN进入细胞后下调转化生长因子β1 m RNA和蛋白的表达。新型阳离子磷酸胆碱基聚合物MPC30-DEA70可以有效负载和运输转化生长因子。

BACKGROUND： Currently, antisense oligonucleotides （AS-ODN） have a good prospect in gene therapy, but AS-ODN with small molecular weight cannot easily enter into the cells, which is susceptible to nuclease degradation. Therefore, there is still a lack of fundamental understanding about how to improve their transfection efficiency, and target-based transferring. OBJECTIVE： To investigate whether a weak cationic and, phosphorylcholine-containing diblock copolymer （MPC30-DEA70） can act as a carrier system to deliver a chemically synthesized transforming growth factor-β1 （TGF-β1） AS-ODN into myocardial cells.METHODS： MPC30-DEA70 was compounded with TGF-β1 AS-ODN at various N/P ratios and the MPC30-DEA7o/TGF-β1 AS-ODN complexes were characterized by DNA electrophoresis. MTT assay was used to observe the biocompatibility. Confocal laser scanning microscope was used to observe the distribution and location of MPC30- DEA70/TGF-β1 AS-ODN in cells. Flow cytometry was used to detect the transfection efficiency and fluorescence intensity of MPC30- DEA70/TGF-β1 AS-ODN in cells. Western blot and RT-PCR methods were employed to measure the expression of TGF-β1 in cells. RESULTS AND CONCLUSION： Cell growth inhibition showed that the MPC30- DEA70 had low cytotoxicity to myocardial cells within the effective transfection dosage range （〈 20 mg/L）. Data from the flow cytometry test indicated a clear trend of increasing transfection efficiency with the increasing of NIP ratios. At high NIP ratios, the expression levels of TGF-β1 mRNA and protein in myocardial cells were significantly lower. This study shows that MPC30-DEA70 can work as an effective transgenic vector in myocardial cells. TGF-β1 AS-ODN can silence the expression of TGF-β1 gene efficiently and specially, and may antagonize TGF-β1-mediated biological function.