人肝癌细胞总rna电转染树突状细胞的体外肿瘤杀伤实验

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1、人肝癌细胞总RNA电转染树突状细胞的体外肿瘤杀伤实验【摘要】目的：探讨人肝癌细胞总RNA电转染人树突状细胞(DendriticCells,DCs)后诱导特异性效应T细胞及其效应T细胞抗肝癌的特异性。方法：通过密度梯度离心法分离人外周血单核细胞，采用细胞因子体外培养诱导其成为未成熟树突状细胞（ImmatureDendriticCell,imDC）和成熟树突状细胞（MatureDendriticCell,mDC）。培养肝癌细胞，Trizol一步法提取人肝癌细胞总RNA。通过电转染法将人肝癌细胞总RNA导入imDC内；

2、观测电转染的转染率；采用免疫细胞化学检测电转染前后的imDC形态及其表面标志；通过混合淋巴细胞实验，获取mDC激活的特异性效应T细胞。用MTT法测定效应T细胞的抗肝癌特异性。结果：电转染方法可将人肝癌细胞总RNA导入imDC。电转染前后imDC分子表达无显著差异，mDC的分子表达与imDC相比发生了变化。转染了人肝癌细胞总RNA的mDC可特异的激活T细胞为效应T细胞。效应T细胞可特异性的杀伤人肝癌细胞，而对不相关的MG-63细胞无特异性的杀伤作用。结论：电转染不影响imDC的形态和原有的蛋白表达；转染了人肝癌细胞

3、总RNA的mDC可特异的激活T细胞为效应T细胞；效应T细胞具有抗人肝癌细胞的特异性。【关键词】树突状细胞肝癌细胞总RNA转染人AbstractObjective:Toinvestiagewhetherthe13electransfectionofhumandendriticcells(DCs)withtotalRNAofhepatocarcinomacellscouldinducethespecificeffectiveTcells,thenanalysisthespecificythateffectiveTce

4、llskilltargethepatocarcinomacells.Methods:MonocytesobtainedformhumanperipheralbloodbydensityguadientcentrifugationareinducedintoimmatureDCsandmatureDCsinthepresenceofcytokinesinvitro.ThetotalRNAofhepatocarcinomacellsareextractedbyTrizolandareeletransfectedint

5、omonocyte-derivedimmatureDCs.Thesuccessrateofelectransfectionismeasured.WeuseimmunocellchemicalstainingtoobservethechangesofimDCs’morphologyandmoleculeexpressionbetweenbeforeandafterelectransfectionofhumandendriticcells(DCs)withtotalRNAofhepatocarcinomacells;

6、weharvesteffectiveTcellsbymixedlymphologyreaction,inwhichgraduallymatureDCsactivateTcells,thenweexaminethespecificitythateffectiveTcellskilltargethepatocarcinomacells.Results:ElectransfectioncanmakeRNAintoimDCsuccessfully.ThereisnosignificantdifferenceinimDCm

7、oleculeexpressionaftertheelectransfection.ThereisdifferentmoleculeexpressionbetweenimDCandmDC.DCswithtotalRNAofhepatocarcinomacellscanactivatespecificallyTcellsintoeffectiveTcellswhichcankillhepatocarcinoma13cellsspecificallywhilenotMG-63cells.Conclusion:Elet

8、ransfectiondoesn’tchangethemorphologyandmoleculeexpression;DCswithtotalRNAofhepatocarcinomacellscanmakeactivationofTcellsandspecificitythattheeffectiveTcellskillhepatocarcinomacellsisexam