ヒト胆道上皮細胞のコラーゲン・ゲル上継代培養による純粋培養法の確立

胶原凝胶传代人胆管上皮细胞纯培养方法的建立

• 批准号: 05770144
• 负责人: 斎藤 勝彦
• 金额: $ 0.58万
• 依托单位: Kanazawa University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Encouragement of Young Scientists (A)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 无数据
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-05770144/
• 关键词: 胆のう; 細胞培養; コラーゲン・ゲル; ヒト

1)ヒト胆嚢粘膜移植片(microexplant)の作製:胆石症の診断で,開腹手術により摘出された胆嚢を,直ちに無菌的に切開し,胆汁を流出させた.粘膜面に付着した胆汁成分を滅菌ガ-ゼで拭き取り,氷冷ハンクス液で充分洗浄した後,肉眼的に著変のない部位を選んで,粟粒大の粘膜片を線維筋層を含まないように剥離切除し,微小移植片(microexplant)を得た.2)ヒト胆嚢粘膜上皮の初代培養系の確立:作製したmicroexplantをI型コラーゲン・ゲル上に静置し,15%牛胎児血清添加培養液(DMEM/HamF12)を重層し,5%CO_2インキュベータ-内に初代培養した.ほとんどのmicroexplantはコラーゲン・ゲルに接着し,培養2日にはヒト胆嚢上皮細胞が周囲のコラーゲン・ゲル上に増殖するのが位相差顕微鏡で確認できた.上皮細胞は培養7日までコラーゲン・ゲル表面を進展増殖し,培養面積が増加したが,以後はコラーゲン・ゲルから剥離し,培養面積は縮小した.移植片が小さいほど間質細胞の増殖は少なく,1〜2週間では上皮の増殖に与える影響は少なかった.上皮細胞の形態は扁平であり,in vivoのごとく円柱状のものはみられなかった.また,粘液産生像は認められなかった.3)培養上皮細胞へのHLA-DRおよび接着分子の誘導:培養4日目に培養液中にインターフェロン-gamma(100ng/ml)を添加して一週間培養し,培養上皮細胞にHLA-DRおよびICAM-Iの発現誘導を試みたが,凍結切片を使用した酵素抗体法では検出できなかった.

1) Preparation of biliary mucosa graft (microexplant): diagnosis of cholelithiasis, open surgery, extraction of bile, sterile incision, bile outflow. The bile components in the mucosa were sterilized and removed. After washing with cold solution, the sites were selected by naked eye, and the micro-explant was obtained. 2) Establishment of primary culture system of bile mucosa: preparation of micro-explant, type I, static culture, 15% fetal bovine serum supplemented culture medium (DMEM/HamF12), heavy layer, 5%CO_2, and primary culture. The microexplant was then cultured for 2 days, and the phase difference between the microexplant and the cultured cells was confirmed by microscopy. Epithelial cells were cultured for 7 days, and the surface of the cells increased, and the culture area increased, and then the cells were peeled off, and the culture area decreased. Grafts are small, stromal cells proliferate, and epithelial cells proliferate and proliferate within 1 to 2 weeks. Epithelial cell morphology is flat,in vivo columnar. 3) Induction of HLA-DR and subsequent molecules in cultured epithelial cells: 4 days after culture, HLA-gamma (100ng/ml) was added to the culture medium during culture, induction of HLA-DR and ICAM-I in cultured epithelial cells was tested, and frozen sections were used.