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跨膜型TNF-α(TM-TNF-α)是激活的单核/巨噬细胞表达在胞膜上的完整蛋白分子。为了确定TM-TNF与细胞膜的关系,本文借助体外转录翻译系统及微粒体膜结构,建立了表达在微粒体膜上的TM-TNF以及无膜的裸26kD TNF。比较这两种体外表达产物对靶细胞的细胞毒效应,结果表明,只有在微粒体存在时所合成的26kD TNF具有细胞毒活性;而无微粒体存在所合成的裸26kD TNF不显示杀瘤效应。进一步用间接免疫荧光技术观察它们与HL60靶细胞胞膜上TNFR的结合情况,结果显示,结合在微粒体膜上的TM-TNF能与TNFR有效结合,而裸26kD TNF与TNFR则不能有效结合。提示TM-TNF只有“锚定”在细胞膜上,才能利于其空间构型的展示,发挥生物学效应;而一旦与膜分离,则不能与TNFR有效地结合,故丧失了其生物学活性。
Transmembrane-type TNF-α (TM-TNF-α) is an intact protein molecule of activated monocytes / macrophages expressed on the cell membrane. To determine the relationship between TM-TNF and cell membrane, TM-TNF expressed on microsomal membranes and non-membrane-bound nude 26 kD TNF were established by in vitro transcriptional translation system and microsomal membrane structure. The cytotoxic effect of these two in vitro expressed products on target cells was compared and the results showed that the 26 kD TNF synthesized only in the presence of microsomes had cytotoxic activity whereas the naked 26 kD TNF synthesized in the absence of microsomes showed no killing effect . Furthermore, indirect immunofluorescence technique was used to observe their binding to TNFR on the membrane of HL60 target cells. The results showed that TM-TNF binding to microsomal membranes could effectively bind to TNFR, while TNF-α and TNF-α could not bind effectively. Tissue TM-TNF only “anchored” in the cell membrane in order to facilitate the display of its spatial configuration, play a biological effect; and once separated from the membrane, it can not effectively bind with TNFR, it lost its biological activity.