Watabe K, Fukuda T, Tanaka J, Honda H, Toyohara K, Sakai O
Division of Neuropathology, Jikei University School of Medicine, Tokyo, Japan.
J Neurosci Res. 1995 Jun 1;41(2):279-90. doi: 10.1002/jnr.490410215.
We established spontaneously immortalized Schwann cell lines from long-term cultures of adult mouse dorsal root ganglia and peripheral nerves. One of the cell lines, designated IMS32, responded to mitogenic stimuli by platelet-derived growth factor (PDGF)-BB, acidic and basic fibroblast growth factors (aFGF, bFGF), and transforming growth factors (TGF)-beta 1 and -beta 2, as determined by bromodeoxyuridine (BrdU) incorporation and double immunofluorescence for S100 and BrdU. Furthermore, conditioned media (CM) obtained from IMS32 cells showed mitogenic activity for both IMS32 cells and long-term cultured Schwann cells. Western blot analysis revealed TGF-beta-like molecule in the CM, and the activity was absorbed with anti-TGF-beta neutralizing antibody. Reverse transcription followed by polymerase chain reaction (RT-PCR) of IMS32 RNA revealed that these cells expressed TGF-beta 1, -beta 2, and -beta 3 transcripts. When rat pheochromocytoma PC12 cells were incubated with the CM, they developed neurite growth. Coculture of PC12 and IMS32 cells also showed neurite growth of PC12 cells. RNA transcripts of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), ciliary neurotrophic factor (CNTF), and glial cell line-derived neurotrophic factor (GDNF) were detected from IMS32 cells by RT-PCR. In these, we sequenced the mouse GDNF cDNA coding region and observed 97% and 90% homologies to corresponding rat and human cDNA sequences, respectively. These results indicate that the immortalized Schwann cell line mitotically responds to various growth factors and secretes autocrine and paracrine growth-promoting activities in vitro.
我们从成年小鼠背根神经节和周围神经的长期培养物中建立了自发永生化的雪旺细胞系。其中一个细胞系命名为IMS32，通过溴脱氧尿苷（BrdU）掺入以及针对S100和BrdU的双重免疫荧光检测发现，它对血小板衍生生长因子（PDGF）-BB、酸性和碱性成纤维细胞生长因子（aFGF、bFGF）以及转化生长因子（TGF）-β1和-β2的促有丝分裂刺激有反应。此外，从IMS32细胞获得的条件培养基（CM）对IMS32细胞和长期培养的雪旺细胞均显示出促有丝分裂活性。蛋白质免疫印迹分析显示CM中存在TGF-β样分子，并且该活性可被抗TGF-β中和抗体吸收。对IMS32 RNA进行逆转录随后进行聚合酶链反应（RT-PCR），结果显示这些细胞表达TGF-β1、-β2和-β3转录本。当大鼠嗜铬细胞瘤PC12细胞与CM一起孵育时，它们会长出神经突。PC12细胞与IMS32细胞共培养也显示出PC12细胞的神经突生长。通过RT-PCR从IMS32细胞中检测到神经生长因子（NGF）、脑源性神经营养因子（BDNF）、神经营养因子-3（NT3）、睫状神经营养因子（CNTF）和胶质细胞系衍生的神经营养因子（GDNF）的RNA转录本。其中，我们对小鼠GDNF cDNA编码区进行了测序，发现其与相应的大鼠和人类cDNA序列分别具有97%和90%的同源性。这些结果表明，永生化的雪旺细胞系在体外对多种生长因子有有丝分裂反应，并分泌自分泌和旁分泌生长促进活性物质。
