针对BAK的siRNA抑制由胶质瘤细胞诱导的树突状细胞凋亡及其机制
摘要
目的树突状细胞与胶质瘤共培养,观察胶质瘤细胞诱导树突状细胞凋亡;观察针对BAK的siRNA对树突状细胞凋亡的影响,检测相应的凋亡基因,探讨其可能凋亡通路,为胶质瘤的免疫治疗提供理论依据。方法树突状细胞与胶质瘤细胞通过孔径0.4μm Trans-well共培养,设置空白DC2.4,为对照组,采用MTT法观察其活性,细胞存活率=实验组吸光度/对照组吸光度×100%。采用脂质体的方法转染表达针对BAK的siRNA序列的质粒(A:229序、B:310序、C:579序、D:NC序)进入树突状细胞,并利用G418筛选出稳定表达株,通过western blot和PCR的方法鉴定出稳定表达有效干扰序列的树突状细胞;稳定表达有效干扰序列的树突状细胞和胶质瘤细胞共培养(b组),设置a组空白DC2.4、c组胶质瘤细胞和DC2.4共培养两个对照组,24h后观察树突状细胞凋亡的改变。通过western blot测定相关基因BAK的表达,观察caspase8,caspase3及胞浆中的细胞色素C的变化,进而推断出凋亡的可能通路。结果胶质瘤细胞与树突状细胞共培养后,与空白树突状细胞组相比树突状细胞的活性降低了,吸光值与空白树突状细胞组相比为0.514±0.031;含有siRNA质粒转染入树突状细胞后,各组BAK的表达均降低,以PGPU6/GFP/Neo-BAK-310组为明显。western blot和RT-PCR检测出310序列BAK的表达降低(western blot:A:0.83、B:0.14、C:0.75、D:0.89、未转染:0.92;RT-PCR:A:0.42、B:0.08、C:0.39、D:0.46、未转染:0.49P＜0.05)。稳定表达有效干扰序列的树突状细胞和胶质瘤细胞共培养后,与c组相比树突状细胞的活性较高,树突状细胞的caspase8表达量无明显差异(a:0.78 b:0.75 c:0.77,P＞0.05),caspase3及胞浆中的Cytc减少(caspase3:a:0.67 b:0.70 c:0.2:Cytc:a:0.70 b:0.71 c:0.17 P＜0.05)。结论胶质瘤细胞与树突状细胞共培养以后,可以诱导树突状细胞凋亡,凋亡途径可能是线粒体途径;关于BAK的有效siRNA序列可抑制胶质瘤细胞诱导的树突状细胞的凋亡,为延长树突状细胞的寿命,提高它的抗原呈递能力提供了理论依据。
Object Dendritic Cells co-culture with glioma,To study the apoptosis of Dendritic Cells that induced by glioma.To study the vector expressing siRNA targeting BAK inhibit the apotosis of Dendritic Cells and to speculate the possible pathway of the apoptosis.Methods The vector that expressing siRNA targeting BAK was transfected into DC by the lipofectamine2000 and the cells stably expressing the siRNA were selected by G418.To choose the high performance vecteor that interference BAK.Then the DC that stably expressing the siRNA were co-culture with glioma and observe the apoptosis of DC.Investing the relate gene for example caspase3 caspase8 and the cytochrome c in endochylema,to speculate the possible pathway of the apoptopsis.Resuits When the DC2.4 co-culture with glioma lead to the apoptosis.When the vector were transfected into DC2.4,the expression of BAK were degraded(western blot:A:0.83,B:0.14,C:0.75,D:0.89,un-transfecte:0.92; RT-PCR.- A:0.42,B:0.08,C:0.39,D:0.46,un-transfecte:0.49 P＜0.05).When the DC2.4 that stably expressing siRNA target BAk co-culture with glioma,the expression of caspase 8 was not change(a:0.78 b:0.75 c:0.77,P＞0.05) to tmexpression siRNA one,but the expression of caspase 3 and the cytochrome C (caspase3:a:0.67 b:0.70 c:0.2;Cytc:a:0.70 b:0.71 c:0.17 P＜0.05)in endochylema was less than the unexpression siRNA one.Conclusion The glioma can induce the apoptosis of DC2.4.The pathway of apoptosis may be the mitochondrial pathway; The vector that expresse siRNA targeting BAk can degrade the apoptosis of DC2.4.
Object Dendritic Cells co-culture with glioma,To study the apoptosis of Dendritic Cells that induced by glioma.To study the vector expressing siRNA targeting BAK inhibit the apotosis of Dendritic Cells and to speculate the possible pathway of the apoptosis.Methods The vector that expressing siRNA targeting BAK was transfected into DC by the lipofectamine2000 and the cells stably expressing the siRNA were selected by G418.To choose the high performance vecteor that interference BAK.Then the DC that stably expressing the siRNA were co-culture with glioma and observe the apoptosis of DC.Investing the relate gene for example caspase3 caspase8 and the cytochrome c in endochylema,to speculate the possible pathway of the apoptopsis.Resuits When the DC2.4 co-culture with glioma lead to the apoptosis.When the vector were transfected into DC2.4,the expression of BAK were degraded(western blot:A:0.83,B:0.14,C:0.75,D:0.89,un-transfecte:0.92; RT-PCR.- A:0.42,B:0.08,C:0.39,D:0.46,un-transfecte:0.49 P＜0.05).When the DC2.4 that stably expressing siRNA target BAk co-culture with glioma,the expression of caspase 8 was not change(a:0.78 b:0.75 c:0.77,P＞0.05) to tmexpression siRNA one,but the expression of caspase 3 and the cytochrome C (caspase3:a:0.67 b:0.70 c:0.2;Cytc:a:0.70 b:0.71 c:0.17 P＜0.05)in endochylema was less than the unexpression siRNA one.Conclusion The glioma can induce the apoptosis of DC2.4.The pathway of apoptosis may be the mitochondrial pathway; The vector that expresse siRNA targeting BAk can degrade the apoptosis of DC2.4.
引文
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