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MOLECULAR CELL BIOLOGICAL SYUDY OF A NOVEL NEUROTROPHIC PROTEIN, PCTF35

新型神经营养蛋白 PCTF35 的分子细胞生物学研究

基本信息

批准号：
11480226
负责人：
UCHIYAMA Yasuo
金额：
$ 9.54万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

项目摘要

To understand in vivo functions of PCTF35, we performed the following experiments using models of brain injuries and primary cultured astrocytes.1) Expression of PCTF35 following brain injuries: For this we prepared the following two animals; (1) Hypoxia-ischemia (H-1) injury: new born rats at P7 were anesthetized and the left common carotid artery was ligated. The animals were returned to the darn for 1 hr and then placed in containers, which were perfused with mixed gases of 8% oxygen and 92% nitrogen and kept at 37℃, for 90 min. They were then returned to the dam until used. (2) Penetrating brain injury: Adult rats (8 weeks of age) were anesthetized and an incision with a 2mm depth and 4mm length was made in the temporal lobe after part of the temporal bone was opened. These animals were perfused from the heart with 4% paraformaldehy, de 3,4,5 and 7 days after the operation and brain tissues were processed for immunohistochemistry. At 3 days, GFAP-immunopositive astrocytes expanded their process and invaded into injured areas, while astogliosis occurred in the areas at 7 days. By double immunostaining, immunoreactivity for PCTF35 from co-localized in the processed of these astrocytes with that for GFAP.2) Expression and secretion of PCTF35 from primary cultured astrocytes: Since PCTF35 was induced in astrocytes appearing in injured areas of brains, we separated astrocytes from brains of rats at P2. PCTF35 was found in the cultured medium of primary astrocytes by Western blot and the amount of the protein was significantly increased in the medium when TGF-β1 was added in cultures. These lines of evidence suggest that PCTF35 is induced in astrocytes of rat brains after H-I and penetrating injuries and this induction is enhanced by TGF-β1.

为了了解PCTF 35的体内功能，我们使用脑损伤模型和原代培养的星形胶质细胞进行了以下实验。1）脑损伤后PCTF 35的表达：为此，我们准备了以下两种动物：（1）缺氧-缺血（H-1）损伤：将P7的新生大鼠麻醉并结扎左颈总动脉。将动物放回母鼠体内1小时，然后置于容器中，容器中灌注8%氧气和92%氮气的混合气体，并在37℃下保持90分钟。然后将动物放回母鼠体内直至使用。(2)穿透性脑损伤：将成年大鼠（8周龄）麻醉，在打开部分颞骨后，在颞叶中形成2 mm深和4 mm长的切口。分别于术后3、4、5、7天用4%多聚甲醛心脏灌流，取脑组织行免疫组织化学染色。在第3天，GFAP免疫阳性星形胶质细胞扩大其进程，并侵入到受伤的地区，而astogliosis发生在该地区在第7天。通过双重免疫染色，PCTF 35的免疫反应性与GFAP的免疫反应性共定位于这些星形胶质细胞的加工过程中。2）从原代培养的星形胶质细胞中表达和分泌PCTF 35：由于PCTF 35在脑损伤区域出现的星形胶质细胞中被诱导，我们在P2时从大鼠脑中分离星形胶质细胞。Western印迹法检测到原代星形胶质细胞培养液中存在PCTF 35蛋白，加入TGF-β1后，培养基中PCTF 35蛋白含量明显增加。这些证据表明，在H-I和穿透性损伤后大鼠脑的星形胶质细胞中诱导PCTF 35，并且这种诱导被TGF-β1增强。