摘要
目的：对一种新发现的凋亡相关基因ＴＦＡＲ１９进行蛋白质水平的促凋亡效应研究，并对其作用机理进行初步的探讨。方法；利用离子交换层析对大肠杆菌表达的人重组ＴＦＡＲ１９蛋白进行纯化，将其加入到培养的白血病细胞株ＨＬ－６０，通过ＤＮＡ片段化、ＰＩ及ＡｎｎｅｋｉｎＶ标记进行流式细胞仪分析，观察ＴＦＡＲ１９蛋白的促凋亡效应。利用ＦＩＴＣ标记ＴＦＡＲ１９蛋白，分析其与细胞的结合及定位。利用Ｃａｓｐａｓｅ－３抑制剂ＤＥＶＤ－ｆｍｋ研究ＴＦＡＲ１９的凋亡信号转导途径。结果：高纯度的重组ＴＦＡＲ１９蛋白剂量依赖性地促进撤除血清的ＨＬ－６０细胞的凋亡，最高可使８２％细胞凋亡，对照为３０％。荧光标记的ＴＦＡＮ１９蛋白能与ＨＬ－６０细胞结合，主要定位于细胞核。ＤＥＶＫ－ｆｍｋ可部分抑制ＴＦＡＲ１９蛋白的促凋亡效应。结论：ＴＦＡＲ１９蛋白能够直接进入ＨＬ－６０细胞，发挥其促进细胞凋亡的效应。这一效应部分地与Ｃａｓｐａｓｅ－３的凋亡执行效应有关。
Objective: To study the apoptosis-accelerating effect of a novel human protein named TFAR19 (TF-1 cells apoptosis relatedprotein 19) whose encoding cDNA has been cloned in the laboratory, and try to find out the mechanism of its effect. Methods: By using ionexchange chromatography , the human recombinant TFAR19 protein was isolated. The parified protein was occulted with HL-60 cells and theapoptotic cells were analyzed by DNA fragmentation, PI and Annexin V-labeled FACS assay. The FTTC labeled TFAR19 protein was used asprobe to observe its localization within the HL-60 cells. For the signal transduction study, a caspase-3 inhibitor DEVD-fmk was used to blockthe TFAR19 effect. Results: The TFAR19 Protein dose-dependently increases the apoptotic HL-60 cells which have been previously serum from culture, up to 82% cells are apoptotic cells comparing with control 30% cells. FITC labeled TFAR19 protein binds with HL-60Cells and mainly locallied within the nucleus. DEVD-fmk pahly inhibits apoptosis-accelerating effect of the TFAR19 protein. Conclusion:TFAR19 protein can directly enter HL-60 cells and exhibits an apoptosis-accelerating effect. Caspase-3 as an apoptosis effector may involve theeffect of TFAR19.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2000年第1期8-11,共4页
Chinese Journal of Immunology
基金
国家自然科学基金!39870427
