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

首先，我们分析了人单核细胞来源的树突状细胞（MoDC）的自然凋亡过程中的生存力和半胱氨酸天冬氨酸蛋白酶2，3，7和8的表达，这些细胞通过IL-1 β，IL-6，TNF α和PGE 2刺激而成熟。MoDC在刺激2d后存活率大于90%，3和4d后降至60-70%，5d后降至50%。凋亡的MoDC百分比在1至4天后持续低于10%，但在6至8天后下降至70-80%。至于共刺激分子如CD 86和HLA-DR抗原的表达，两者在2天后上调，但在6天或更长时间后，发现MoDC的2个亚群：一个保持这两种分子的高表达，另一个减少这些分子。在上述的caspase中，只有caspase-2在MoDC的凋亡过程中表达上调。首先，我们尝试用脂质体转染法转染GAPDH siRNA，以寻找最佳的转染试剂。我们使用了TransIT-TKO[○！R]（Mirus），siPortNeoFX[○！R]和siPortAmine[○！R]（Ambion），Dharmafect1，2，3，4[○！R]（Dharmacon）和Lipofectamine 2000 [○！R]（Invitrogen）。在这些试剂中，Lipofectamine 2000 [○！R]表现出最好的结果，最大抑制44%。我们还尝试了使用Nucleofector^<TM>II（Amaxa）的电泳方法，导致最大70%的抑制。我们现在正试图将caspase 2-siRNA转染到MoDC中，并检查caspase 2-siRNA是否可以延长成熟MoDC的活力。

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.