酪丝亮肽荧光标记物的合成及其在肿瘤治疗靶点研究中的应用  

Synthesis of Tyroserleutide Fluorescent Analogue and Its Application on The Target Research of Antitumor Therapy

作　　者：简序[1] 傅正[1] 张燕玲[2] 韩丽霞[2] 朱志峰[1] 徐琼[1] 王莉[1] 周春雷[1] 石琳熙[1] 刘俊燕[1] 陆融[1] 姚智[1] 

机构地区：[1]天津医科大学免疫学教研室 [2]天津医科大学医学检验系,天津300203

出　　处：《生物化学与生物物理进展》2008年第10期1161-1167,共7页Progress In Biochemistry and Biophysics

基　　金：国家高技术研究发展计划(863)(2004AA2Z3170,2005AA2Z3D40);国家重点基础研究发展计划(2003CCA04300);教育部重点项目(03007)资助~~

摘　　要：酪丝亮肽是一种具有抗肿瘤活性的小分子三肽,它可以诱导造成肝癌细胞发生凋亡坏死,从而杀伤肿瘤细胞,但是酪丝亮肽在肝癌细胞的亚细胞定位尚不十分明确.为了达到对酪丝亮肽进行示踪进而观察其亚细胞定位的目的,使用荧光物质(5(6)-羧基四甲基罗丹明琥珀酰亚胺酯,5(6)-TAMRASE)对酪丝亮肽进行了标记,应用非变性聚丙烯酰胺凝胶电泳、毛细管电泳和荧光分光光度法对标记酪丝亮肽进行纯化和鉴定.并在激光扫描共聚焦显微镜下观察了荧光标记酪丝亮肽在人肝癌BEL-7402细胞中的分布.结果显示,合成的酪丝亮肽荧光标记物性质稳定,标记的酪丝亮肽在人肝癌BEL-7402细胞的胞浆中呈聚集分布.Tyroserleutide （YSL） is a new anticancer polypeptide developed recently. It can induce hepatocellular carcinoma cells apoptosis and necrosis. However, the subcellular targets of YSL in the hepatocellular carcinoma cells are not known very well. Therefore, a fluorescent bioconjugate of 5-（and-6）-carboxytetramethylrhodamine, succinimidyl ester （5 （6）-TAMRASE）-YSL）was synthesized and purified by native polyacrylamide gel electrophoresis and capillary electrophoresis. After stability was assessed by capillary electrophoresis and fluorescent spectrophotometric assay, concentration of this conjugate was quantitated by fluorescent spectrophotometric assay. Then subcellular distribution of 5（6）-TAMRASE labeled YSL were investigated by laser scanning confocal microscopy to gain a better understanding of the anticancer mechanism of YSL in vitro. The experimental results showed that the 5（6）-TAMRASE can conjugate with YSL stably. When incubated with human hepatocellular carcinoma BEL-7402 cells, 5 （6）-TAMRASE labeled YSL could entered the cells in 15 min, and mainly concentrated in the cytoplasm of BEL-7402 cells. Then the fluorescent intensity of 5（6）-TAMRASE labeled YSL which indicated the concentration of them reach the maximum until incubated with the cells for 2 h. After that it began to go down. The 5（6）-TAMRASE could also enter the hepatocellular carcinoma BEL-7402 cells, but it distributed in the whole cell and the intensities had no decrease within 3 h. The results suggest that the distribution pattern of fluorescent labeling YSL is different from that of 5 （6）-TAMRASE. The subcellular distribution pattern of 5 （6）-TAMRASE labeled YSL was ascribed to YSL and could represent that of YSL. In conclusion, YSL located at the cytoplasm of hepatocellular carcinoma BEL-7402 cells and distributed intensively.

关 键 词：酪丝亮肽 肝癌 荧光标记 激光扫描共聚焦显微镜 

分 类 号：R341.6[医药卫生—基础医学] R979.1