Optimizing method of pancreatic cancer MiaPaCa-2 cells total RNA-transfected dendrtic cells
10.3760/cma.j.issn.1674-1935.2011.01.008
- VernacularTitle:胰腺癌MiaPaCa-2细胞总RNA体外转染树突细胞的方法探讨
- Author:
Jiang CHEN
;
Xiaozhong GUO
- Publication Type:Journal Article
- Keywords:
Pancreatic neoplasms;
Dendritic cells;
RNA Transfection
- From:
Chinese Journal of Pancreatology
2011;11(1):20-23
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the best method of transfecting total RNA extracted from pancreatic cancer MiaPaCa-2 cells into dendrtic cells (DCs). Methods DCs were cultured from peripheral blood mononuclear cells induced by rhGM-CSF, rhIL-4 and TNF-α. Morphology of DCs was observed. Flow cytometry was used to detect the mature DCs specific surface markers:CD40, HLA-DR, CD83, CD86. Mixed lymphocyte (MLR) was used to determine the ability of DCs to stimulate allogeneic T cell proliferation.Liposomal transfection, 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 stimulate 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 (3 1. 68 ± 7.25) and passive transfection method (18.53 ± 3.26) . 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.
