Gobé G C, Halliday J, Dunn I S, Harmon B V, Schoch E, Khan S M, Allan D J
Department of Pathology, University of Queensland Medical School, Herston, Brisbane, Australia.
Exp Cell Res. 1997 Sep 15;235(2):354-61. doi: 10.1006/excr.1997.3682.
An in vivo neonatal rat kidney model was used to study an association between expression and localization of the retinoblastoma tumor-suppressor gene (Rb), or its protein product (pRb), and localization of radiation-induced apoptosis. The rat kidney has two distinct zones of differentiation at birth-an outer nephrogenic zone, in which cells are undifferentiated and new nephrons are forming, and a differentiated zone internal to this zone that has essentially the adult kidney form. At 6 h after radiation (5 Gy), high levels of relatively synchronous apoptosis are induced in the nephrogenic zone, with little effect on the differentiated zone, and proliferation in the nephrogenic zone is almost totally inhibited by radiation treatment, again with little effect in the differentiated area. We have used our knowledge of this model to analyze control (sham-treated) and irradiated renal tissue for Rb mRNA transcript levels and localization (Northern blot and in situ hybridization (ISH)), pRb expression (Western blot and immunolocalization), apoptosis and mitosis (light and electron microscopy, and DNA gel electrophoresis for apoptosis), and cells in S-phase ([3H]thymidine uptake and autoradiography). Northern blots showed no detectable alteration in Rb transcript levels between control and irradiated tissues, whereas Western blots indicated increased expression of pRb in protein extracted from irradiated kidney compared with controls. ISH confirmed that Rb transcripts were not substantially altered in the nephrogenic and differentiated zones in control versus irradiated renal tissue. Immunolocalization of pRb demonstrated little effect in the differentiated zone, but in the nephrogenic zone pRb expression was increased, especially the S-shaped prenephrons, and was also found in many, but not all, apoptotic cells in this zone. The results link radiation-induced apoptosis and increased pRb expression in a zone of the neonatal kidney having a low level of cell differentiation.
采用体内新生大鼠肾脏模型，研究视网膜母细胞瘤抑癌基因（Rb）或其蛋白产物（pRb）的表达与定位，以及辐射诱导的细胞凋亡定位之间的关联。大鼠肾脏在出生时有两个不同的分化区域——外部的肾发生区，其中细胞未分化且新的肾单位正在形成，以及该区域内部的分化区，其基本具有成年肾脏的形态。在辐射（5 Gy）后6小时，肾发生区诱导出高水平的相对同步的细胞凋亡，对分化区影响很小，并且辐射处理几乎完全抑制了肾发生区的增殖，对分化区的影响同样很小。我们利用对该模型的了解，分析对照（假处理）和辐照后的肾组织中的Rb mRNA转录水平和定位（Northern印迹和原位杂交（ISH））、pRb表达（Western印迹和免疫定位）、细胞凋亡和有丝分裂（光学和电子显微镜检查，以及用于细胞凋亡的DNA凝胶电泳），以及处于S期的细胞（[3H]胸苷摄取和放射自显影）。Northern印迹显示对照组织和辐照组织之间Rb转录水平无明显变化，而Western印迹表明，与对照相比，从辐照肾脏提取的蛋白质中pRb表达增加。ISH证实，对照肾组织与辐照肾组织相比，肾发生区和分化区的Rb转录本没有实质性改变。pRb的免疫定位显示对分化区影响很小，但在肾发生区pRb表达增加，尤其是S形前肾单位，并且在该区域许多（但不是全部）凋亡细胞中也有发现。结果表明，在新生肾脏细胞分化水平较低的区域，辐射诱导的细胞凋亡与pRb表达增加有关。
