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P2-1-111 マウス脳における神経上皮細胞と上衣細胞に発現する新たな細胞膜表面抗原の同定 Identification of a cell membrane surface antigen, INCA antigen, expressed on the epithelial and ependymal cells in mouse brain ○小谷政晴1, , 佐藤安訓2, 上野明道2, 伊藤頼位3, 伊藤康一4, 今田正人5 ○Masaharu Kotani1, Yasunori Sato2, Akemichi Ueno2, Toshinori Ito3, Kouichi Itoh4, Masato Imada5 奥羽大・薬・分子細胞生物1, 奥羽大・薬・衛化2, 奥羽大・薬・英教3, 徳島文理大・香川薬・分子薬理4, 日大・医・機能形態5 Dept Mol Cell Biol, Ohu Univ, Koriyama1, Dept Hygi Chem, Ohu Univ, Koriyama2, Dept Eng Lang Tech, Ohu Univ, Koriyama3, Dept Mol Pharm, Kagawa Camp, Tokushima Bunri Univ, Shoto4, Dept Funct Morpho, Nihon Univ Sch Med, Tokyo5 It is unclear whether neural stem cells (NSCs) in the lateral ventricles (LVs) of adult vertebrate brain are subventricular zone (SVZ) astrocytes or ventricular zone (VZ) ependymal cells. To approach this problem, we tried the generation of a novel monoclonal antibody (mAb) which reacts with neuroepithelial cells in embryonic brains and got one mAb, INCA mAb, which fulfills this purpose, by immunizing rats with the membrane fraction of embryonic day 13.5 (E13.5) telencephalons. In this talk, we present the characterization of INCA mAb and the identification of INCA mAb recognition antigens (INCA antigens) positive cells (INCA+ cells). INCA mAb reacted with VZ and SVZ in embryinic brains and VZ in 6-week-old brains (adult brains). INCA antigens were 40 and 42 kDa proteins present in the membrane fractions of embryonic and adult brains and neurospheres. The INCA mAb recoginition epitope on INCA antigens was an O-linked carbohydorate moiety. Immunohistochemical analysis with existing biomarkers revealed that INCA antigens were expressed on neuroepithelial and ependymal cells, but not on neurons or glial cells. Additionally, INCA antigens were expressed in fetal neurospheres, prepared from E13.5 telencephalons, and in adult neurospheres, prepared from surrounding area of the LVs in adult brains, and the proportions of INCA+ cells in these neurospheres were approximately 76.35% and 65.08%, respectively. Moreover, a group of neural cells differentiated from these neurospheres expressed β-catenin and S100β as well as INCA antigens, which strongly suggests that they are ependymal cells. Collectively, it was concluded that INCA antigens were constitutively expressed in a linage from neuroepithelial cells to ependymal cells, suggesting the possibility that INCA+ cells had the capability as NSCs.	P2-1-112 532 nm 低出力レーザー照射はin vivoとin vitroにおいて神経幹細胞/前駆細胞の増殖を促進する 532 nm low-power laser can promote the proliferaion of neural stem/progenitor cells in vivo and in vitro ○福崎由美1, 菅原はるな1, 川井秀樹1, 山之端万里2, 木暮信一1 ○Yumi Fukuzaki1, Haruna Sugawara1, Hideki Kawai1, Banri Yamanoha2, Shinichi Kogure1 創価大学大学院 工学研究科 生命情報工学専攻1, 創価大学 工学部 環境共生工学科2 Bioinfo Major, Grad Sch of Engr, Soka Univ, Tokyo1, Dep of Environ, Fac of Engr, Soka Univ, Tokyo2 Introduction It has been found that the neural stem/progenitor cells (NSPCs) exist in the adult brain. However, the self-renewal of them is extremely at low rate, resulting in small presence of NSPCs in the brain. We have already demonstrated that 532 nm low-power laser irradiation (LLI) can promote cell proliferation of human-derived glioblastoma through the Akt protein activation. The present study was designed to reveal whether 532 nm LLI could influence NSPCs proliferation in vivo as well as in vitro or not, with an aim to develop a regenerative therapy for intractable brain diseases. Methods All experiments were performed according to the bioethical guidelines. <In vitro> NSPCs from mouse embryos were cultured in N2 medium. Neurospheres were moved to 384 well plate (1 sphere/well). Experimental groups were irradiated for 20, 40 and 60 min by a diode laser (Nd:YVO4; CW; 532 nm; 60 mW; 7.1 mm2). The wells were photographed at pre-, post-irradiation, 24h after LLI. <In vivo> Transient mild cerebral ischemia was induced by bilateral common carotid artery occlusion for 10 min using adult mice. One week after, the skull was exposed under anesthesia and auditory cortical area (A1) in the left hemisphere was irradiated by the same laser, while the right was not as a control. Next day, under deep anesthesia, the animal was perfused with 4% PFA, then brain was removed and cut for immunofluorescent staining with Ki67 and GAD or for TTC staining. Result The size ratio of each neurosphere standardized with the initial condition showed a significant promotion by LLI with an irradiation time-dependency: 60 min LLI was the most effective (t-test, p<0.05). In addition, the number of Ki 67 positive cells increased in layer 1 to 6 of left A1 area. TTC staining showed almost no cell death in both hemispheres. Conclusion From the results obtained in both experiments, we conclude that 532 nm LLI (60 mW) could promote the self-renewal of NSPCs.
P2-1-113 発達中のニワトリ嗅覚系におけるソマトスタチン発現抑制の効果 Somatostatin affects development of the GnRH neuron system in the chick olfactory-forebrain axis ○村上志津子1, 内山安男1 ○Shizuko Murakami1, Yasuo Uchiyama1 順天堂大・医・ 神経生物学・形態学1 Dept Cell Biol and Neurosci, Juntendo Univ Sch of Med1 Transient expression of somatostatin (SS) has been found in the olfactory epithelium, the olfactory nerve fibers and the cells migrating from the olfactory epithelium of chick embryos. The times of appearance and distribution of SS immunoreactive (-ir) neural elements are similar to those of GnRH neurons which originate in the olfactory placode and then migrate into the forebrain. To examine the possible role of SS in the development of the olfactory-forebrain axis, the expression of SS mRNA was specifically knocked down in the olfactory epithelial cells by small interfering RNA (siRNA). Chick embryos at embryonic day (E) 3.5 were electroporated into the olfactory pit region to transfer siRNA oligonucleotides against mRNA of chick somatostatin and analyzed at E6.5. The pCAGGS vector containing the EGFP was coelectroporated. Nonsilencing siRNA was used as a negative control. On the operated side in the embryos electroporated with siRNA for SS mRNA, SS expression was remarkably reduced in the olfactory epithelium. In 2 out of 4 embryos analyzed, the normally developed olfactory nerve was observed on the electroporated (EP) side. In another two embryos, the olfactory nerve bundle was slightly thinner than that detected in the contralateral non-electroporated (NEP) side. No detectable disruption of the olfactory nerve was observed in the control siRNA-treated embryos. Although GnRH neurons were observed in their migration pathway of the nasal-forebrain region, the relative proportion of GnRH neurons in the EP side to that in the NEP side was low in the SS siRNA-treated embryos, compared with that of control siRNA-treated embryos. Moreover, the proportion of GnRH neurons in the forebrain on the EP side tended to be reduced in the SS siRNA-treated embryos when compared with it in the NEP side. These results indicate that siRNA for SS mRNA introduced into the developing olfactory epithelium considerably decreased the number of migrating GnRH neurons.

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