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Treatment of arthritic model mice using hematopoietic stem cells simultaneously transfected with genes for anti-inflammatory cytokines and chemokine receptors

使用同时转染抗炎细胞因子和趋化因子受体基因的造血干细胞治疗关节炎模型小鼠

基本信息

批准号：
13670470
负责人：
MINOTA Seiji
金额：
$ 0.32万
依托单位：
Jichi Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

The purpose of this study is to utilize hematopoietic stem cells as a vehicle of the anti-inflammatory cytokines in the arthritic model mice. T cells derived from these hematopoietic stem cells must go preferentially to the inflammed joints in order to take better effect. Two putative genes were chosen : chemokine receptor (CCR4) and IL-10. The former is to direct T cells to the inflammed joints and the latter is for anti-inflammatory effect. These genes need to be transfected simultaneously in a single hematopoietic stem cell.As a preliminary experiment, peripheral T cells were used instead of hematopoietic stem cells from bone marrow, because transfection efficacy is much better for peripheral T cells than for hematopoietic stem cells. Gene for green fluorescent protein (GFP) was incorporated into retrovirus and was transfected to peripheral T cells as a reporter gene along with chemokine receptor or IL-10. Transfection efficacy was increased to "30% from 10% by repeating transfection processes on the fibronectin-coated plate including anti-CD3 and anti-CD28 antibodies.Arthritic model mice were made by subcutaneous injections into DBA/1 mice of bovine type II collagen emulsified with Freund's complete adjuvant, booted with the same antigen in incomplete adjuvant 3 weeks afer the first immunization. By the biginning of the fourth week, many joints from immunized mice began to swell and arthritis became evident. However, the severity and the frequency of the artificial arthritis made by this method were quite diverse and, therefore, evaluation of the effectiveness of the treatment is quite difficult. Experimentation to overcome this issue is now undergoing.

本研究的目的是利用造血干细胞作为关节炎模型小鼠抗炎细胞因子的载体。从这些造血干细胞衍生的T细胞必须优先进入炎症关节，以发挥更好的作用。选择两个推定的基因：趋化因子受体（CCR 4）和IL-10。前者是将T细胞导向炎症关节，后者是抗炎作用。这些基因需要在单个造血干细胞中同时转染，作为初步实验，使用外周T细胞代替来自骨髓的造血干细胞，因为外周T细胞的转染效率比造血干细胞好得多。将绿色荧光蛋白（GFP）的基因整合到逆转录病毒中，并作为报告基因与趋化因子受体或IL-10一起沿着至外周T细胞。通过在包括抗-CD 3和抗-CD 28抗体的纤连蛋白包被的板上重复转染过程，转染效率从10%增加到30%。通过向DBA/1小鼠皮下注射用弗氏完全佐剂乳化的牛II型胶原蛋白，在第一次免疫后3周用在不完全佐剂中的相同抗原引导，制备关节炎模型小鼠。到第四周的第二天，免疫小鼠的许多关节开始肿胀，关节炎变得明显。然而，通过这种方法制造的人工关节炎的严重程度和频率是相当多样化的，因此，评估治疗的有效性是相当困难的。目前正在进行克服这一问题的试验。