生体の組織を構成している分化機能を持つ細胞は200種を超えると思われる. これら多様な細胞機能を解明し, 生体組織の機能特性を再構築するためには, 個々の分化機能を保持した細胞を不死化することが必要となる. 筆者らは, 温度感受性SV40 T-抗原遺伝子導入トランスジェニックマウス, およびラットを作出し, 多様な組織から分化機能を保持した不死化細胞株を樹立する方法を確立した. 筆者らは, 造血幹細胞を支持する骨髄の間質細胞株を多数不死化し, 骨髄造血微小環境のin vitro再構成を目指した研究を進め, これら間質細胞株が, 細胞間相互作用により培養系で造血細胞の特異的支持機能を示すとともに, 骨芽細胞, 内皮細胞, 筋細胞, 脂肪細胞, 平滑筋細胞など多様に分化できる間葉系幹細胞としての性質も持っていることを明らかにした. これら不死化した機能細胞株は, 今後, 再生医学的研究とともに, 医薬品スクリーニングシステムや毒性評価システムの構築, 新規生理活性物質の探索などへの創薬研究への利用も期待できる.

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Retinal degenerative disease such as retinitis pigmentosa and age-related macular degeneration causes visual defects with the progress in their diseases. In case of retinitis pigmentosa a large number of mutations in genes that involved in phototransduction pathway have been identified. However pharmacological treatment is presently unavailable for these diseases. Several strategies such as retinal pigment epithelial cell transplantation, growth factor application, vitamin supplementation or gene therapy are under examination for preventing the photoreceptor cell death. Among these strategies, some growth factors are commonly effective on preventing photoreceptor cell death even the mutated genes cause that photoreceptor cell death is different. Is it possible to prevent photoreceptor cell death and to maintain the visual function by continuously supplying growth factors to photoreceptor cells? Which methodology is better to supply growth factor continuously, gene transfer to retina or transplantation? We describe here the approach to prevent photoreceptor cell death by using gene transfected iris pigment epithelial cell transplantation.

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膵臓の発生分化, β細胞の再生の機構を理解することは, 糖尿病の治療の観点上, 大変重要である. 膵臓はadultではほとんど分裂しないが, なんらかの原因でβ細胞が損傷されると, 膵再生が起きることが観察されていたので, 分裂能を持つ“膵幹細胞”の存在が考えられていた. ここでは, 膵臓の発生分化, 細胞系譜の解析, 膵臓の前駆細胞についての最近の知見を解説する. そして, 膵臓の前駆細胞あるいはES細胞を用いた試験管内におけるβ細胞の再生系について紹介し, 膵臓再生への展望について概説する.

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ヒトゲノムプロジェクトが完遂し, ゲノム創薬がクローズアップされている. 条件的不死化細胞は, いわゆる“ポストゲノム”の時代において, ハイスループットスクリーニングなどにより, 迅速に大量の化合物をスクリーニングする際のツールとしてもきわめて有用であり, 創薬のシーズ探索のツールとして大きな貢献が期待される. 条件的不死化細胞株化のための手法の一つとして, 温度感受性SV40 T遺伝子を導入した動物を用いる方法がある. 本手法により樹立された細胞株は, 培養温度が33℃の条件では増殖するが, 39℃にシフトさせると増殖を停止し分化するという特性を持つ. したがって本手法においては, 第一に, これまで株化が困難であった組織や, 微量にしか採取できない細胞の, 許容培養温度条件下での増殖が可能となり, より広範な種類の細胞の株化が期待される. 第二に, 培養温度をコントロールするだけで細胞分化を誘導できることから, 分化に伴って発現する遺伝子プロファイルを調べたり, その機能解析などへの応用も可能である. また, さらに, 特定の遺伝子を欠損した動物と交配して得られた動物から同様に細胞株が樹立できれば, 欠損遺伝子の生理機能の解析手段となりうる. 実際, ビタミンD受容体(VDR)が欠損した細胞株を作出し, 1,25(OH)₂D₃は, VDR依存性に骨髄間質細胞の脂肪細胞への分化を抑制していることを明らかにすることができた. 今後, 本手法が創薬をはじめ医薬品開発に活用されることを期待したい.

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Elucidating the molecular mechanism of the brain harrier transport is a key step toward successful drug delivery to the brain. In this review, we focused on the usefulness and the limitations of the newly developed in vitro model. To overcome several limitations of classical in vitro model, conditionally immortalized mouse (TM-BBB) and rat (TR-BBB) brain capillary endothelial cell lines and a choroid plexus epithelial cell line (TR-CSFB) were established from the temperature sensitive SU 40 large T antigen gene transgenic animals. mRNA and protein expression of GLUT1 in TR-BBB was 100 folds greater than that of primary culture, Several known transporter genes were identified for TR-BBB or TM-BBB, including organic anion transporter 3 (OAT 3) and organic anion transporting polypeptide 2 (oatp 2). Employing TM-BBB, new brain barrier functions have been clarified such as creatine transporter (CRT) and GABA transporter (GAT 2/BGT-1). TR-BBB has been applied for the study of expressional regulation of transporters at the BBB, e. g., L-proline and glycine transporter (ATA 2) and taurine transporter (TAUT). Moreover, one of the most important subjects for the in vitro BBB model is to clarify the expression and regulation of the tight junction proteins at the BBB. Co-culture systems among TR-BBB, conditionally immortalized astrocyte (TR-AST) and pericyte (TR-PCT) cell lines were developed and revealed that the expression of occludin, a tight-junction component, was induced by soluble factors secreted from TR-AST and TR-PCT. For the brain drug delivery research, the new in vitro model will provide rational and efficient strategies.

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Syncytiotrophoblasts, which form a continuous barrier between the maternal and fetal circulation, play an essential role in restriction of drug delivery through the blood-placental barrier (BPB). In the BPB many influx and efflux transporters for substrates such as neurotransmitters, neuroactive steroids, and nucleosides may play in the development of the fetus and fetal brain. However, the functions of these transporters in BPB, and the molecular and cellular aspects of these transporters remain unclear. The human term placenta and human chorionic carcinoma cell lines, such as BeWo and JAR cells have been mainly used for analysis of transporter and hormone secretion in the placenta. However it is difficult to confirm in vivo evidence using these conventional cell lines. In order to investigate the relationship between in vitro and in vivo results, cell lines from animals as an in vitro model are needed. We have established new syncytiotrophoblast cell lines (TR-TBTs) from the recently developed transgenic rat harboring temperature-sensitive simian virus 40 large T-antigen gene (Tg-rat). TR-TBTs have temperature-sensitive cell growth due to the expression of is SV 40 large T-antigen. These cell lines had a syncytium-like morphology, could be prepared as monolayers, expressed cytokeratins, rat syncytiotrophoblast markers, and in vivo influx and efflux transporters such as oatp2, TauT, GAT-2/BGT-1, and mdr 1 a. Moreover, TR-TBTs exhibited apical or basal GLUT1. localizations and apical GLUT3 localizations. Thus, these results suggest that TR-TBTs could be very useful tool to clarify the BPB functions, including transporter genes and their regulation systems.

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