载HCPT纳米粒的制备及其体外抗肿瘤活性研究

目的:制备载羟基喜树碱(HCPT)的PLGA-hyd-PEG-FA纳米粒(HCPT@PLGA-hyd-PEG-FA),并对其体外抗肿瘤活性进行研究。方法:采用乳化溶剂挥发法制备HCPT@PLGA-hyd-PEG-FA,通过单因素试验考察超声功率、聚合物浓度、PVA浓度、水相和油相体积比及投药量对纳米粒粒径的影响;采用zeta电位及激光粒度分析仪测定纳米粒的粒径及zeta电位,用透射电镜(TEM)观察其形态;采用透析法评价HCPT@PLGA-hyd-PEG-FA的体外释药特性;采用MTT法测定HCPT@PLGA-hyd-PEG-FA对HepG2细胞的细胞毒性。结果:HCPT@PLGA-hyd-PEG-FA平均粒径约为109±3 nm,zeta电位为-11.57 mV,载药量为5.6%,TEM显示其为球形;体外释药结果表明HCPT@PLGA-hyd-PEG-FA对HCPT的释放具有p H值依赖性;HCPT和HCPT@PLGA-hyd-PEG-FA的IC50值分别为474.6 ng/mL和286.0 ng/mL。结论:HCPT@PLGA-hyd-PEG-FA体外释药性能良好,HCPT@PLGA-hyd-PEG-FA的细胞毒性明显大于游离的HCPT,值得进一步研究。     

Role of Bmbuffy in hydroxycamptothecine-induced apoptosis in BmN-SWU1 cells of the silkworm, Bombyx mori

Bcl-2 family proteins have been reported previously to play important roles in the mitochondrial apoptotic pathway. Particularly, Bmbuffy has been identified as a key homologue of Bcl-2 in silkworm; however, its exact function is unknown. In this study, we investigated the role of Bmbuffy in hydroxycamptothecine (HCPT)-induced apoptosis of BmN-SWU1 cells. By conducting confocal microscopy studies, we found that Bmbuffy is located on the outer membrane of mitochondria and endoplasmic reticulum (ER). Furthermore, we discovered that the hydrophobic transmembrane domain at the COOH terminus is a putative anchor for the subcellular localization of Bmbuffy. Overexpression of Bmbuffy inhibited cytochrome c release, activation of caspase-3 and cell apoptosis, while RNAi-mediated silencing of Bmbuffy promoted apoptosis. In the absence of a hydrophobic membrane anchor, we revealed that Bmbuffy is unable to block apoptosis. These results indicate that Bmbuffy acts as an anti-apoptotic protein, located on the mitochondrial outer membrane and is involved in the mitochondrial apoptotic pathway. Moreover, in HCPT-induced apoptosis, we showed that the translocation of endogenous Bmp53 from the nucleus to the mitochondria is a slow and progressive process, followed by cytochrome c release. This suggests that mitochondrial Bmp53 accumulation may contribute to membrane permeability. The co-localization of Bmp53 and Bmbuffy suggests the interaction of the two proteins, which was further confirmed by Co-IP assay. In addition, overexpression of Bmp53 increased cytochrome c release and the cell apoptotic rate, whereas Bmbuffy overexpression blocked these. All the data suggest that Bmbuffy functions as an anti-apoptotic protein and interacts with Bmp53 in HCPT-induced apoptosis of silkworm cells.     

羟基喜树碱纳米颗粒超声反溶剂法制备工艺及表征

为提高羟基喜树碱的水溶性及其制剂在体内稳定性,运用超声反溶剂法对羟基喜树碱进行初级颗粒制备。本文根据羟基喜树碱在不同有机溶剂中的溶解度与其在去离子水中的溶解度的不同进行对比,对各项指标进行检测。筛选出制备羟基喜树碱初级颗粒的最佳条件为:HCPT-DMSO浓度为5 mg·mL-1、H2O∶DMSO=1∶1、H2O滴速为4 mL·min-1,超声时间为10 min。对羟基喜树碱初级纳米颗粒进行检测,SEM扫描结果显示其形貌规则,粒径为746 nm,与羟基喜树碱原粉颗粒相比,粒径明显减小并在其水溶液中分布更为均匀。     

羟喜树碱结合肽的研究

酯化脱水法制备生物素化羟喜树碱,使用Resourse 15反相柱纯化,液质联用分析确证。磺酰罗丹明B染色法鉴定生物素化羟喜树碱的抗癌活性。采用噬菌体展示技术筛选得到14条羟喜树碱结合肽。构建并优化了羟喜树碱结构模型。构建并通过分子动力学优化了羟喜树碱结合肽结构模型。使用分子对接验证并分析了羟喜树碱结合肽与羟喜树碱的结合作用。