ヒト誘導型制御性T細胞培養液中に存在する細胞増殖抑制因子の同定と応用に関する研究

人诱导调节性T细胞培养物中细胞生长抑制因子的鉴定及应用研究

• 批准号: 18790931
• 负责人: 伊藤 淳
• 金额: $ 2.24万
• 依托单位: Kyoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Young Scientists (B)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18790931/
• 关键词: 制御性T細胞; 免疫抑制剤

(1)ラパマイシン誘導ヒト制御性T細胞の産生する細胞増殖抑制因子の分離精製およびアミノ酸配列:健常人ボランティアより得た末梢血より,密度勾配遠心法により単核球を分離後,磁気ビーズ法を用いてCD4+T細胞を分離し,ラパマイシン及びIL-2存在下,抗ヒトCD3/CD28結合ビーズを用いて連続刺激することにより,細胞を培養増殖させた.細胞数が目的の量に達した段階で,細胞を洗浄し,ラパマイシンおよびIL-2を除き,無血清培養液を用いて,4日間培養後,培養液を回収した.得られた培養液を添加することにより,ヒト末梢血単核球の抗ヒトCD3抗体刺激による細胞増殖活性は,濃度依存的に抑制された.得られたラパマイシン誘導制御性T細胞の培養液を限外ろ過/遠心式フィルターにより濃縮し,ゲルろ過カラムクロマトグラフィー法により培養液中の細胞増殖抑制因子を分離した結果,細胞増殖抑制活性は,分子量約10kDの分画中に認められた.さらに,SDS-PAGE法により分離後,質量分析装置により解析した結果,3種類のタンパク質が同定された.(2)培養上清を用いた増殖抑制因子の生物活性の検討:細胞増殖制活性を持つ培養上清を濃縮後,マウス同種異系皮膚移植モデルに投与し,移植拒絶抑制効果に関して検討した.マウス皮膚移植モデルは,BalbヌードマウスにC57BL/6マウスの皮膚を移植し,Balb/cマウスより得たT細胞を移入することにより,拒絶を誘導した.濃縮培養培養上清投与群の皮膚移植拒絶期間は,コントロール群と比較し場合に,統計的有意な差は認められなかった.

The main results are as follows: (1) the cell colonization inhibitory factors of T cells are isolated from normal people. In normal people, the peripheral blood is obtained. After the nuclear spheres are separated by the density matching method, the cells are separated by the magnetic resonance method, and the cells are separated by the magnetic resonance method in the presence of CD4+T and IL-2. The combination of anti-tumor CD3/CD28 and anti-virus stimulates the growth of anti-tumor cells with the combination of anti-virus and anti-virus. Objective to measure the number of cells in each section, wash the cells, remove the IL-2, and use the serum-free culture solution. After 4 days of incubation, the culture solution will return to the temperature. The culture liquid was added to the culture solution, the peripheral blood nuclear globule, the anti-CD3 antibody, the cellular colonization activity and the degree-dependent inhibition activity were obtained. The recombinant T-cell culture solution was used to determine the molecular weight of T-cell culture. The results showed that the cell proliferation inhibitory factor in the culture solution was isolated, the cell colonization inhibitory activity was determined, and the molecular weight was about 50% in 10kD separation. After the quantitative analysis was performed by SDS-PAGE method, the results were analyzed by quantitative analysis device. (2) the culture supernatant was cultured with anti-proliferative inhibitory factor bioactivity assay. After the culture supernatant was incubated with the culture supernatant, the transplantation of the same kind of pedicled skin was successful, and the transplantation was rejected. The skin transplant was not allowed, the Balb graft was not allowed, the C57BL/6 was grafted, the Balb/c cell was moved into the skin graft, and the guide was rejected. In the period of rejection of skin transplantation, there was a significant difference between the two groups in the period of rejection.

项目成果

制御性T細胞による拒絶反応抑制-免疫抑制剤からの解放-

调节性 T 细胞抑制排斥反应 - 免受免疫抑制剂的影响 -

• 发表时间: 2007
• 期刊: 日本臨牀 65巻・3号
• 作者: 小柴 貴明;伊藤 淳;李 穎;呉 艶玲;竹村 摩美;坂口 志文
• 通讯作者: 坂口 志文