MOLECULAR CELL BIOLOGICAL SYUDY OF A NOVEL NEUROTROPHIC PROTEIN, PCTF35

为了了解PCTF35在体内的功能，我们利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PCTF35的表达：为此我们制备了2只动物；(1)缺氧缺血（H-1）损伤：在P7时麻醉新生大鼠，结扎左颈总动脉。实验结束后，将实验动物送回试验室1小时，放入容器中，充注8%氧气和92%氮气的混合气体，在37℃下保存90分钟。然后，它们被送回大坝，直到使用。(2)穿透性脑损伤：成年大鼠（8周龄）麻醉后，在颞叶上切开部分颞骨，切口深度2mm，长度4mm。术后第3、4、5、7天从心脏灌注4%多聚甲醛，脑组织进行免疫组化处理。在第3天，gfap免疫阳性的星形胶质细胞扩大其进程并侵入损伤区域，而在第7天发生星形胶质细胞形成。2)原代培养的星形胶质细胞中PCTF35的表达和分泌：由于PCTF35是在脑损伤区出现的星形胶质细胞中诱导产生的，我们在P2时将星形胶质细胞从大鼠的大脑中分离出来。Western blot法在原代星形胶质细胞培养液中发现PCTF35，在培养液中加入TGF-β1后，该蛋白在培养基中的表达量显著增加。这些证据表明，在H-I和穿透性损伤后，PCTF35在大鼠脑星形胶质细胞中被诱导，TGF-β1增强了这种诱导作用。

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.