Investigation for the methods of enhanced tumor vaccination using apoptosis-resistant dendritic cells manipulated by transfection of small-interfering RNAs responsible for the apoptosis of dendritic cells

研究通过转染负责树突状细胞凋亡的小干扰RNA来操纵抗凋亡树突状细胞增强肿瘤疫苗接种的方法

• 批准号: 17591155
• 负责人: NAKAGAWA Satoshi
• 金额: $ 2.3万
• 依托单位: TOHOKU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17591155/
• 关键词: dendritic cells; apoptosis; small interfering RNA

First we analyzed the viability and the expression of caspases 2, 3, 7 and 8 during the natural apoptotic course of human monocyte-derived dendritic cells (MoDC) which were matured by stimulation with IL-1beta, IL-6, TNFalpha, and PGE2. The viability of MoDC was more than 90% after 2 days of stimulation and decreased to 60-70% after 3 and 4 days, and finally fell into 50% after 5 days. The percentage of apoptotic MoDC was sustained less than 10% after 1 to 4 days, but decreased into 70-80% after 6 to 8 days. As for the expression of the costimulatory molecules such as CD86 and HLA-DR antigen, both were upregulated after 2 days but after 6 days and longer, 2 subpopulations of MoDC were found : one keeping high expression of both molecules, the other reducing these molecules. Among the caspases described above, only caspase-2 was found to be upregulated during the course of the apoptotic process of MoDC.Next we performed to transfect small interfering RNA (siRNA) into human MoDC. First we tried to transfect GAPDH siRNA by lipofection methods to search the best transfection reagents. We used TransIT-TKO【○!R】(Mirus), siPortNeoFX【○!R】 and siPortAmine【○!R】 (Ambion), Dharmafect1, 2, 3, 4【○!R】 (Dharmacon), and Lipofectamine2000【○!R】 (Invitrogen). Among these reagents, Lipofectamine2000【○!R】 performed the best results, maximally 44% of the inhibition. We also tried electrophoration methods using Nucleofector^<TM> II (Amaxa) resulting in maximally 70% of inhibition. We are now trying to transfect caspase2-siRNA into MoDC and examining whether caspase2-siRNA can prolong the viability of mature MoDC.

首先，我们分析了在il -1 β、IL-6、TNFalpha和PGE2刺激下成熟的人单核细胞源性树突状细胞（MoDC）自然凋亡过程中caspases 2、3、7和8的活性和表达。MoDC的存活率在刺激2天后达到90%以上，3、4天后降至60-70%，5天后降至50%。1 ~ 4 d后，凋亡的MoDC比例持续低于10%，6 ~ 8 d后下降至70 ~ 80%。对于CD86和HLA-DR抗原等共刺激分子的表达，在2天后均上调，但在6天后或更长时间内，发现MoDC的两个亚群：一个保持这两个分子的高表达，另一个降低这两个分子的表达。在上述的caspase中，只有caspase-2在MoDC的凋亡过程中被发现上调。接下来，我们将小干扰RNA （siRNA）转染到人MoDC中。首先，我们尝试用脂质体法转染GAPDH siRNA，寻找最佳的转染试剂。我们使用了TransIT-TKO【〇！】（负），siPortNeoFX【〇！R】和siPortAmine【〇！（Ambion）， dharmafec1, 2, 3, 4【〇！】R】（Dharmacon）， Lipofectamine2000【〇！（Invitrogen）。其中Lipofectamine2000【〇！R】的抑制效果最好，最高可达44%。我们还尝试了使用核因子^<TM> II （Amaxa）的电泳方法，最大抑制率为70%。我们正在尝试将caspase2-siRNA转染到MoDC中，并检测caspase2-siRNA是否可以延长成熟MoDC的生存能力。