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，我们准备了以下两种动物：(1)缺氧缺血(H-1)损伤：新生P7龄大鼠麻醉，结扎左侧颈总动脉。将动物放回笼子中1h，然后放入容器中，容器中注入8%氧气和92%氮气的混合气体，保持在37℃，90min。然后，它们被送回大坝，直到被使用。(2)穿透性脑损伤：成年大鼠(8周龄)麻醉后，在部分颞骨切开后，在颞叶做一个深2 mm、长4 mm的切口。分别于术后第3、4、5、7天从心脏灌流4%甲醛，取脑组织进行免疫组织化学染色。3d时，GFAP免疫阳性星形胶质细胞突起扩大并侵入损伤区，7d时损伤区星形胶质形成。2)原代培养星形胶质细胞PCTF35的表达和分泌：由于PCTF35是在出现在脑损伤区域的星形胶质细胞中诱导表达的，所以我们在P2分离了大鼠脑内的星形胶质细胞和星形胶质细胞。Western印迹显示原代培养的星形胶质细胞培养上清液中有PCTF35表达，加入转化生长因子-β-1后，培养上清液中的蛋白含量明显增加。这些证据表明，大鼠脑缺血和穿透伤后星形胶质细胞表达PCTF35，而转化生长因子-β-1能增强这种诱导作用。

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.