Studies on the Proteins of Thymocytes Induced by X ray and Cause Apoptosis.

经低剂量X射线照射的大鼠胸腺细胞在数小时内发生间期细胞死亡。这种间期死亡被称为程序性或生理性细胞死亡，细胞凋亡是一种特有的、精心安排的自我破坏形式，它被蛋白质合成的抑制剂如放线菌素D或放线菌素D所阻止。在本项目中，我们试图纯化和鉴定与X射线照射的胸腺细胞凋亡有关的RNA和蛋白质。我们从X射线照射后4h的大鼠胸腺细胞的mRNA中制备了c DNA文库。用X射线照射和对照胸腺细胞RNA的差减方法筛选诱导RNA文库。共获得31个克隆。将其插入pGEM载体中，用RNA聚合酶合成mRNA，注射到非洲爪哇卵母细胞中。3个RNA能降解卵母细胞，但不能引起大鼠胸腺细胞的间期死亡。为了筛选引发胸腺细胞死亡的RNA，我们试图找出将RNA注入胸腺细胞的好方法，并确定细胞凋亡的特征。电穿孔被发现是最有效的将放射性RNA轻松地整合到相对大量的胸腺细胞中。作为细胞凋亡的指标，微量凝胶电泳法观察染色体破坏较常用的染色排斥法或细胞体积缩小法更可靠。用这些方法筛选RNA的工作正在进行中。在双向电泳法寻找诱导蛋白时，我们在染色凝胶上发现了5个斑点，在放射自显影上发现了4个斑点，这些斑点在X射线照射的样品中比对照组强。对这些蛋白质的结构分析目前正在进行中。

Rat thymocytes irradiated with a low dose of X rays undergo interphase cell death within several hours. This interphase death is known as programmed or physiological cell death, apoptosis, a characteristic and well orchestrated form of selfdestruction, which is prevented by the inhibitors proteinsy nthesis such as cycloheximide or actinomycin D. In this project, we tried to purify and identify the RNA's and proteins which are responsible for the apoptosis of thymocytes irradiated with X ray. We prepared cDNA library from the mRNA of rat thymocytes 4 hours after X ray irradiation. The library was screened for the induced RNA by the subtraction method of X ray irradiated and control thymocyte RNA. 31 clones were obtained. From the cDNA inserted into pGEM plasmid, mRNA was synthesized with RNA polymerase and injected into Xenopus oocytes. 3 RNA's were found to degradate oocytes, but they did not cause interphase death of rat thymocytes. To screen for the RNA which trigers the thymocyte death, we tried to find out the good methods to inject RNA into thymocytes and to decisive feature of apoptosis. The electroporation was found to be most efficient to incorporate radioactive RNA into relatively large number of thymocytes readily. As a criterion the apoptosis, the observation of chromosome destruction on minigel electrophorasis was more reliable than the commonly used dye-exclusion or cell-volume reduction. The screening of RNA by these methods are now in progress. In searching for the induced proteins by two-demensional electrophoresis, we found 5 spots on dye-stained gels and 4 spots on autoradiogram which are stronger for the X ray irradiated samples than in control. Structural analysis of these proteins are now in progress.