胰腺癌MiaPaCa-2细胞总RNA体外转染树突细胞的方法探讨

Optimizing method of pancreatic cancer MiaPaCa-2 cells total RNA-transfected dendrtic cells

目的探讨将胰腺癌MiaPaCa-2细胞总RNA转染树突细胞（DCs）最优化的方法。方法以rhGM—CSF、rhIL-4和TNF-α联合诱导外周血单核细胞以获得DCs，观察DCs的形态变化，流式细胞术检测DCs表面标志CD40、HLA—DR、CD83和CD86表达，混合淋巴细胞反应（MLR）测定DCs刺激异体T细胞增殖的能力。采用脂质体转染、电穿孔及被动转染三种方法将MiaPaCa-2总RNA转染DCs，应用实时定量PCR法检测MUC1 mRNA表达，MTF法测定DCs存活率。结果所获细胞具有典型的成熟DCs形态特征，CD40、HLA—DR、CD83和CD86阳性表达率分别为34．3％、50．2％、89．2％和73．6％，对同种异体T淋巴细胞具有极强的刺激增殖作用。电穿孔法DCs转染48h后，DCs的MUC1 mRNA表达量为45．39±9．33，明显高于脂质体法的31．68±7．25和被动转染法的18．53±3．26；DCs存活率为（80．36±2．43）％，较被动转染法的（91．48±5．42）％略低，但高于脂质体法的（67．44±2．51）％，且基本稳定在80％左右。结论采用电穿法将胰腺癌MiaPaCa－2细胞总RNA体外转染DCs的效率较高，且较安全。

Objective To investigate the best method of transfeeting total RNA extracted from pancreatic cancer MiaPaCa-2 cells into dendrtic cells （DCs）. Methods DCs were cultured from peripheral blood mononuelear cells induced by rhGM-CSF, rhIL-4 and TNF-α Morphology of DCs was observed. Flow eytometry was used to detect the mature DCs specific surface markers： CD40, HLA-DR, CD83, CDs6. Mixed lymphocyte （MLR） was used to determine the ability of DCs to stimulate allogeneie T cell proliferation. Liposomal transfeetion, electroporation method and passive transfection was used to transfect MiaPaCa-2 cell total RNA into DCs, Real time RT-PCR and MTT assay was used to determine the expression of MUC1 mRNA and the survival rate of the RNA transfected DCs. Results The cells acquired showed typical DCs morphology, the positive rate of CD40, HLA DR, CD83 and CD86 were 34.3% ,50.2% ,89.2% and 73.6% , and they showed a strong ability to stinmlate allogeneic T cell proliferation. 48 h after transfection with MiaPaCa-2 cells total RNA by using electroporation, the MUC1 mRNA amount （45.39 ± 9.33） in DCs was higher than that of liposomes method （31.68 ±7.25 ） and passive transfection method （ 18.53 ± 3.26 ） o DCs survival rate was （ 80.36 ±2.43 ） % by using electroporation, which was relatively lower than （91.48 ± 5.42 ） % by using passive transfection method, but higher than （67.44 ± 2.51 ） % by using liposomes method, and it was stabilized around 80%. Conclusions Transfecting total RNA extracted from pancreatic cancer MiaPaCa-2 cells into DCs with electroporation is efficient and safe.