Wang Ye, Mi Shuang-Li, Lou Mei-Yan, Gao Yin, Chen Zhang-Liang, An Cheng-Cai
National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, China.
Front Biosci. 2005 Sep 1;10:2279-84. doi: 10.2741/1697.
Trichosanthin (TCS) is a ribosome-inactivating protein (RIP) which can inhibit the growth of human choriocarcinoma (JAR) cells. There are no clear mechanisms to discover the interaction pathway and cytotoxicity of TCS in JAR cells. In this paper, we showed the distribution and transport of endogenously expressed TCS in JAR cells. Enhanced Green Fluorescence Protein (EGFP), fused with TCS, was applied as a reporter to track the behavior of TCS in JAR cells. Firstly, we investigated the expression stability of EGFP and physiological effects on JAR cells. A stable cell line expressing EGFP was created, which could reproduce and express EGFP even if transplanted into nude mice. Based on the proved stability and feasibility of EGFP in cultured cells and in vivo, the fusion gene of EGFP and TCS was constructed and transfected into JAR cells by liposome. The fluorescence microscopy showed that TCS-EGFP fusion gene was expressed in JAR cells in 24 to 48 hours and the fluorescence spread in cytoplasm mainly and in nucleus partially, which could trace the distribution and transport of TCS-EGFP in JAR cells. Most of fluorescent cells died after 48 hours for the cytotoxicity of expressed TCS-EGFP. These results first reported a stable expression and tracing method by EGFP in JAR cells, and provided theoretical basis to apply TCS in cancer therapy.
天花粉蛋白（TCS）是一种核糖体失活蛋白（RIP），能够抑制人绒毛膜癌细胞（JAR）的生长。目前尚不清楚TCS在JAR细胞中的相互作用途径和细胞毒性的具体机制。在本文中，我们展示了内源性表达的TCS在JAR细胞中的分布和转运情况。将与TCS融合的增强型绿色荧光蛋白（EGFP）用作报告基因，以追踪TCS在JAR细胞中的行为。首先，我们研究了EGFP的表达稳定性及其对JAR细胞的生理影响。构建了一个表达EGFP的稳定细胞系，即使移植到裸鼠体内，该细胞系仍能繁殖并表达EGFP。基于EGFP在培养细胞和体内已证实的稳定性和可行性，构建了EGFP与TCS的融合基因，并通过脂质体转染到JAR细胞中。荧光显微镜观察显示，TCS-EGFP融合基因在24至48小时内在JAR细胞中表达，荧光主要在细胞质中扩散，部分在细胞核中扩散，这可以追踪TCS-EGFP在JAR细胞中的分布和转运。由于表达的TCS-EGFP具有细胞毒性，大多数荧光细胞在48小时后死亡。这些结果首次报道了EGFP在JAR细胞中的稳定表达和追踪方法，为TCS在癌症治疗中的应用提供了理论依据。
