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

此前，我们在猴脑中发现了血小板衍生生长因子- b链（PDGF-B）及其受体的表达。在这个项目中，我们证明了，1)PDGF-B在正常大鼠脑中的表达：在正常和成熟大鼠脑中，我们通过原位杂交组织化学确定了PDGF-B基因在神经元中的定位。northern blot结果显示，这些转录本分别为3.5 kb和2.6 kb。通过检测脑内pdgf - b相关的有丝分裂活性证实了该蛋白的表达。2)与脑损伤和脑移植相关的PDGF-B表达调控：局灶性脑缺血、缺氧/缺血性损伤等病变附近神经元中PDGF-B表达快速、短暂性升高。在大鼠嗅球移植研究中，PDGF-B在嗅神经束向周围神经细胞辐射的单个轴突附近表达。这些结果表明PDGF-B具有保护危险神经元和重塑神经纤维网络的作用，并对培养的神经细胞具有神经营养作用。局灶性脑缺血后，短暂性PDGF-B上调后，神经元、反应性胶质细胞、巨噬细胞等细胞中PDGF-B受体表达增加。脑刺伤后巨噬细胞中PDGF-B的表达与反应性星形胶质细胞形成相关。这些结果在局灶性缺血性损伤和刀伤中得到进一步提示PDGF-B在损伤脑愈合过程中的作用。3)从脑中克隆PDGF-B基因：在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中克隆了其基因。PDGF-B基因的长未翻译5'端序列已被证明可抑制其蛋白翻译。在本研究中，脑损伤后增加的短转录本被确定为截断形式，缺少其5'端非翻译序列。揭示了PDGF-B蛋白表达的新调控机制，即诱导基因表达截断，在5′端非翻译区缺乏翻译抑制序列，为PDGF-B蛋白翻译提供模板。4)综上所述，我们确定了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，从脑中克隆基因还揭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