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为了探讨在胞浆中表达的PrP的理化特征以及对细胞活性的影响,我们构建了胞浆型PrP(CytoPrP)真核表达质粒,瞬时转染人神经母细胞瘤SH-SY5Y细胞,通过蛋白酶敏感性实验检测CytoPrP及其蛋白酶抗性,利用MTT和Trypan Blue细胞计数检测CytoPrP的细胞毒性作用。结果显示,CytoPrP在胞浆内的存在受蛋白酶抑制剂的控制;与野生型PrP相比,CytoPrP具有相对较强的蛋白酶K(PK)抗性。MTT和细胞计数实验均显示,Cyto-PrP的存在可诱导明显的细胞毒性效应,而CytoPrP的细胞毒性作用受蛋白酶抑制剂含量的影响,呈剂量依赖关系。上述结果为研究CytoPrP在朊病毒病的发病机制中的意义提供了一定的科学数据。
To investigate the physical and chemical characteristics of PrP expressed in the cytoplasm and its effect on the cell viability, we constructed a cytoplasmic PrP (CytoPrP) eukaryotic expression plasmid and transiently transfected the human neuroblastoma SH-SY5Y cells through protease-sensitive CytoPrP and its protease resistance were tested in sexual experiments. CytoPrP cytotoxicity was measured using MTT and Trypan Blue cell counts. The results show that the cytoplasmic presence of CytoPrP is controlled by a protease inhibitor; CytoPrP has a relatively strong resistance to proteinase K (PK) compared to wild-type PrP. MTT and cell counting experiments showed that Cyto-PrP can induce significant cytotoxic effect, while CytoPrP cytotoxicity was affected by protease inhibitor in a dose-dependent manner. The above results provide some scientific data for studying the significance of CytoPrP in the pathogenesis of prion diseases.