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

通过基因克隆和原位杂交分析脑缺血中的血小板衍生生长因子。

• 批准号: 06670223
• 负责人: SASAHARA Masakiyo
• 金额: $ 1.41万
• 依托单位: Shiga University of Medical Science
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06670223/
• 关键词: cloning; in situ hybridization; neuron; platelet-derived growth factor; gene; transplantation; gliosis; ischemia; 脳; マクロファージ; 外傷

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

此前，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。在这里，在这个项目中，我们证明了，1）正常大鼠大脑中的PDGF-B表达：在正常和成熟的大鼠大脑中，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。如 Northern blot 所示，这些转录本几乎为 3.5 和 2.6 kb。通过检测大脑中 PDGF-B 相关的有丝分裂活性证实了该蛋白的表达。 2）与脑损伤和移植相关的PDGF-B表达的调节：在局灶性脑缺血和缺氧/缺血性损伤等病灶附近的神经元中诱导PDGF-B表达快速且短暂的增加。在大鼠嗅球移植研究中，在单个轴突从嗅神经束到周围神经毡的辐射附近观察到PDGF-B表达。在脑损伤和移植模型中获得的这些发现表明，PDGF-B 应在保护处于危险中的神经元和重塑神经纤维网络中发挥作用，并且之前已证明其对培养的神经元细胞具有神经营养活性。局灶性脑缺血后，神经元、反应性神经胶质细胞和巨噬细胞等细胞中 PDGF-B 受体表达增加，随后出现短暂的 PDGF-B 上调。脑刺伤后，巨噬细胞中PDGF-B的表达与反应性星形胶质细胞增生相关。在局灶性缺血性损伤和刺伤中获得的这些结果进一步表明PDGF-B在受伤脑的愈合过程中的作用。 3)从脑克隆PDGF-B基因：局灶性脑缺血后立即观察到短形式的PDGF-B基因的快速诱导。我们通过锚定PCR克隆技术成功地从大脑中克隆了其基因。 PDGF-B 基因的长非翻译 5' 端序列已被证明可抑制其蛋白质翻译。在本研究中，脑损伤后增加的短转录物被确定为截短形式，缺乏其5'端非翻译序列。揭示了新的蛋白表达调控机制，即诱导截短的基因表达，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) 总之，我们确定了神经元中PDGF-B基因的表达及其与损伤和移植相关的表达。此外，从大脑中克隆的基因证明了蛋白质表达的新调控机制。较少的

项目成果

12) Chang Y.Mary et al.: "Inhibition of hypercholesterolemia-induced atherosclerosis in the nonhuman primate by probucol : II.cellular composition and proliferation" Arterioscler, Thromb.Vasc.Biol.15. 1631-1640 (1995)

12) Chang Y.Mary 等人：“普罗布考对非人灵长类动物高胆固醇血症诱导的动脉粥样硬化的抑制：II.细胞组成和增殖”Arterioscler，Thromb.Vasc.Biol.15。

Sasahara Masakiyo,: "Analysis of secretion and expression of platelet-derived growth factor in cultured endothelial cells from stroke-prone spontaneously hypertensive rat," J.Cell Biochem.,suppl.18A. 272 (1994)

Sasahara Masakiyo，：“来自易发生中风的自发性高血压大鼠的培养内皮细胞中血小板衍生生长因子的分泌和表达的分析”，J.Cell Biochem.，suppl.18A。

IIHARA Koji: "Coordinate expression of platelet-derived growth factor-B chain and β-receptor in focal ischemia of rat brain," Stroke. 25. 267- (1994)

IIHARA Koji：“大鼠脑局灶性缺血中血小板衍生生长因子-B 链和 β-受体的协调表达”，《中风》25. 267- (1994)。

笹原正清: "成熟ラット脳における血小板由来増殖因子B-鎖発現の解析" 日本神経病理学会会誌. 35. 201- (1994)

Masakiyo Sasahara：“成年大鼠大脑中血小板衍生生长因子 B 链表达的分析”日本神经病理学会杂志 35. 201-（1994）。

Sasahara Masakiyo: "Inhibition of hyperecholesterolemia-induced atherosclecrosis in nonhuman primate by producol : I. Is the extent of atherosclerosis related to resistance of LDL to oxidation?" J. Clin. Invest.94. 155-164 (1994)

Sasahara Masakiyo：“producol 对非人灵长类动物高胆固醇血症诱导的动脉粥样硬化的抑制作用：I. 动脉粥样硬化的程度与 LDL 对氧化的抵抗力有关吗？”