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

用低剂量X射线照射的大鼠胸腺细胞在数小时内经历间期细胞死亡。这种间期死亡被称为程序性或生理性细胞死亡、细胞凋亡、一种特征性的和精心策划的自我毁灭形式，其被蛋白质合成抑制剂如放线菌酮或放线菌素D阻止。本课题试图从X射线照射的胸腺细胞中分离纯化与凋亡有关的RNA和蛋白质。以X射线照射后4h的大鼠胸腺细胞mRNA为材料，制备了cDNA文库。用X射线照射胸腺细胞RNA和对照胸腺细胞RNA差减法筛选诱导后的RNA。共获得31个克隆。将cDNA插入pGEM质粒，用RNA聚合酶合成mRNA，注射非洲爪蟾卵母细胞。发现3种RNA能降解卵母细胞，但不引起大鼠胸腺细胞的间期死亡。为了筛选出能引起胸腺细胞凋亡的RNA，我们试图寻找合适的RNA注射方法，并研究胸腺细胞凋亡的决定性特征。发现电穿孔最有效地将放射性RNA容易地掺入相对大量的胸腺细胞中。用微凝胶电泳观察染色体的破坏情况作为判断细胞凋亡的指标，比常用的染料排斥法或细胞体积缩小法更为可靠。通过这些方法筛选RNA的工作正在进行中。在双向电泳中，我们在染色凝胶上发现了5个点，在放射自显影图上发现了4个点，这些点在X射线照射样品中比对照样品中更强。这些蛋白质的结构分析正在进行中。

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.