Analysis of platelet-derived growth factor in cerebral ischemia, by gene cloning and by in situ hybridization.

在此之前，我们在猴脑中发现了血小板源性生长因子B链（PDGF-B）及其受体的表达。1）PDGF-B在正常大鼠脑组织中的表达：在正常和成熟大鼠脑组织中，我们用原位杂交组织化学方法检测了PDGF-B基因在神经元中的定位。北方杂交结果表明，这些转录本大小约为3.5和2.6kb。通过检测脑中PDGF-B相关的促有丝分裂活性来证实蛋白质表达。2)与脑损伤和移植相关的PDGF-B表达的调节：在损伤附近的神经元中，如局灶性脑缺血和缺氧/缺血损伤，诱导PDGF-B表达的快速和短暂的增加。在大鼠嗅球的移植研究中，PDGF-B的表达被观察到在从嗅神经束到周围神经元的单个轴突的辐射附近。这些发现在脑损伤中获得， 关于我们 实验和移植模型表明，PDGF-B在保护濒危神经元和重塑神经纤维网络中发挥作用，以及先前证明的对培养的神经元细胞的神经营养活性。在局灶性脑缺血后，短暂的PDGF-B上调之后是细胞如神经元、反应性胶质细胞和巨噬细胞中PDGF-b受体表达的增加。脑刺伤后，巨噬细胞内PDGF-B的表达与反应性星形胶质细胞增生相关，这一结果进一步提示了PDGF-B在脑损伤愈合过程中的作用。3)从脑中克隆PDGF-B基因：局灶性脑缺血后立即观察到短型PDGF-B基因的快速诱导。我们利用锚定PCR克隆技术成功地从脑组织中克隆了其基因。PDGF-B基因的长的非翻译的5 '端序列已经显示抑制其蛋白质翻译。在本研究中，脑损伤后增加的短转录本被确定为截短形式，缺少其5 '端非翻译序列。新的蛋白质表达调控机制被证实，即诱导截短的基因表达，使5 '端非翻译区缺少翻译抑制序列，为PDGF-B蛋白翻译提供模板。4)总之，我们已经确定了PDGF-B基因在神经元中的表达及其与损伤和移植相关的表达。此外，从脑中克隆的基因展示了新的蛋白表达调控机制。少

Previously, we found platelet-derived growth factor-B chain (PDGF-B) and its receptor expression in monkey brain. Here, in this project, we demonstrated, 1) PDGF-B expression in normal rat brain : In normal and mature rat brain, we have determined the localization of PDGF-B gene in neurons by in situ hybridization histochemistry. These transcripts were almost 3.5 and 2.6 kb, as shown by northern blot. The protein expression was confirmed by the detection of PDGF-B-related mitogenic activity in the brain. 2) Regulation of PDGF-B expression related to cerebral insults and transplantation : Rapid and transient increase of PDGF-B expression was induced in the neurons in the vicinity of the lesions, such as focal cerebral ischemia and hypoxic/ischemic injury. In the transplantation study of rat olfactory bulb, PDGF-B expression was observed in a close proximity to the radiation of individual axon from olfactory nerve bundles to surrounding neuropil. These findings obtained in cerebral injur … More ies and transplantation model suggest that PDGF-B should play a role in the protection of neurons at risk, and in the remodeling of neural fiber network, together with previously demonstrated its neurotrophic activity to cultured neuronal cells. Following focal cerebral ischemia, transient PDGF-B upregulation was followed by increase of PDGF-b receptor expression in cells such as neurons, reactive glial cells and macrophages. After cerebral stab wound, the expression of PDGF-B in macrophages correlated with reactive astrogliosis.These results obtained in focal ischemic injury and stab wound further suggest the role of PDGF-B in the healing process of the injured brain. 3) Cloning of the PDGF-B gene from brain : Immediately after focal cerebral ischemia, rapid induction of PDGF-B gene of short form was observed. We successfully cloned its gene from brain by use of anchored PCR cloning. Long untranslated 5'-end sequence of PDGF-B gene has been shown to inhibit its protein translation. In the present study, short transcripts which increased after cerebral injury was determined as truncated form, lacking their 5'-end untranslated sequence. Novel regulatory mechanism of protein expression was demonstrated, that is, induced truncated gene expression, lacking translation inhibitory sequence in 5'-end untranslated region, supply template for PDGF-B protein translation. 4) In summary, we have determined PDGF-B gene expression in neurons and its expression related to insults and transplantation. Further, gene cloning from brain demonstrated novel regulatory mechanism for the protein expression. Less