CD1d基因稳定转染Panc-1细胞系的建立
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摘要
实验采用PCR法获得CD1d基因片段,将其插入慢病毒pReceiver-Lv201载体(带GFP荧光)中获得EX-S0249-LV201重组质粒,经转染试剂EndoFectin-Lenti转染到293T细胞中获得慢病毒LP-S0249-LV201,用包装获得的慢病毒感染胰腺癌Panc-1细胞,在不同时间段用倒置荧光显微镜观察绿色荧光,并用反转录酶聚合酶链反应(RT-PCR)和Western blot方法验证获得的表达细胞株.实验结果显示经RT-PCR和Western blotting检测证实慢病毒LP-S0249-LV201转染至Panc-1细胞株后,该细胞株表达CD1d基因和蛋白,成功建立了稳定表达CD1d基因的Panc-1细胞系,为进一步研究CD1d基因在胰腺癌免疫基因治疗中的作用奠定基础.
The PCR method is adopted in this experiment to obtain CD1d gene fragment,which is then inserted into the lentiviral pReceiver-Lv201( with GFP fluorescence) to obtain the EX-S0249-LV201 recombinant plasmid. It is transfected thereafter into the 293T cells with the help of reagent EndoFectin-Lenti to get lentiviral LP-S0249-LV201,which is then infected into pancreatic Panc-1 cells. The inverted fluorescent microscope is employed to observe green fluorescent at different times. In the meanwhile,the reverse transcriptase polymerase chain reaction( RT-PCR) and Western blot are used to validate the expression of cell lines. The experimental results show that,according to RTPCR and Western blotting,the Panc-1 cell line expresses CD1d gene and protein after being transfected with lentiviral LP-S0249-LV201. The Panc-1 cell line which can steadily express CD1d is successfully established. This paper lays the foundation for the further research of CD1d gene in the Pancreatic cancer immuno-gene therapy.
The PCR method is adopted in this experiment to obtain CD1d gene fragment,which is then inserted into the lentiviral pReceiver-Lv201( with GFP fluorescence) to obtain the EX-S0249-LV201 recombinant plasmid. It is transfected thereafter into the 293T cells with the help of reagent EndoFectin-Lenti to get lentiviral LP-S0249-LV201,which is then infected into pancreatic Panc-1 cells. The inverted fluorescent microscope is employed to observe green fluorescent at different times. In the meanwhile,the reverse transcriptase polymerase chain reaction( RT-PCR) and Western blot are used to validate the expression of cell lines. The experimental results show that,according to RTPCR and Western blotting,the Panc-1 cell line expresses CD1d gene and protein after being transfected with lentiviral LP-S0249-LV201. The Panc-1 cell line which can steadily express CD1d is successfully established. This paper lays the foundation for the further research of CD1d gene in the Pancreatic cancer immuno-gene therapy.
引文
[1]Rossjohn J,Pellicci DG,Patel O,et al.Recognition of CD1d-restricted antigens by natural killer T cells[J].Nat Rev Immunol,2012,12(12):845-57.
[2]Ning B T,Song H,Yang S L,et al.Establishment of a transgenic cell line with stable expression of human CD14[J].Journal of Zhejiang University(Medical Science),2012,41(5):506-11.
[3]Ning B T,Tang Y M.Establishment of the cell line,HeLa-CD14,transfected with the human CD14 gene[J].Oncol Lett,2012,3(4):871-874.
[4]Zhu Y,Cheng M,Lu N et al.Construction of NK4 gene lentiviral vector and its expression in bone mesenchymal stem cells[J].Journal of Biomedical Engineering,2011,28(5):976-81.
[5]Doi R.Pancreatic tumor:progress in diagnosis and treatment.Topics:III.Pancreatic endocrine tumor;3.Multiple endocrine neoplasia[J].Nihon Naika Gakkai Zasshi,2012,101(1):116-9.
[6]Shi S,Yao W,Xu J,et al.Combinational therapy:new hope for pancreatic cancer?[J].Cancer Lett,2012,317(2):127-35.
[7]Chang D,Osman K,Connolly J,et al.Sustained expansion of NKT cells and antigen-specific T cells after injection of alphagalactosylceramide loaded mature dendritic cells in cancer patients[J].Exp Med,2005,201(9):1503-1517.
[8]Macho Fernandez E,Chang J,Fontaine J,et al.Activation of invariant Natural Killer T lymphocytes in response to theα-galactosylceramide analogue KRN7000 encapsulated in PLGA-based nanoparticles and microparticles[J].Int J Pharm,2012,423(1):45-54.
[9]Kuylenstierna C,Snyder-Cappione JE,Loo CP,et al.NK cells and CD1d-restricted NKT cells respond in different ways with divergent kinetics to IL-2 treatment in primary HIV-1 infection[J].Scand J Immunol,2011,73(2):141-146.
[10]Raghuraman G,Geng Y,Wang C R,IFN-b-Mediated Up-Regulation of CD1d in Bacteria-Inf-ected APCs[J].J Immunol,2006,177(11):7841-7848.
[11]师义,王昆华,刘为军,等.CD1d分子研究进展[J].广东医学,2012 33(11):1678-1680.
[12]Hix L M,Shi Y H,Brutkiewicz R R,et al.CD1d-expressing breast cancer cells modulate NKT cell-mediated antitumor immunity in a murine model of breast cancer metastasis[J].PLoS One,2011,6(6):e20702.
[13]Sun X Z,Liu G H,Wang Z Q,et al.Over-expression of VEGF165 in the adipose tissue-derived stem cells via the lentiviral vector[J].Chin Med J(Engl),2011,124(19):3093-7.
[14]Giry-Laterrière M,Verhoeyen E,Salmon P.Lentiviral vectors[J].Methods Mol Biol,2011,737:183-209.
[15]Xing H Y,Liu T,Gong Y P.Study of transfection of human embryonic stem cell with green fluorescent protein mediated by lentiviral vectors[J].Chinese Journal of Hematology,2011,32(5):349-51.
[2]Ning B T,Song H,Yang S L,et al.Establishment of a transgenic cell line with stable expression of human CD14[J].Journal of Zhejiang University(Medical Science),2012,41(5):506-11.
[3]Ning B T,Tang Y M.Establishment of the cell line,HeLa-CD14,transfected with the human CD14 gene[J].Oncol Lett,2012,3(4):871-874.
[4]Zhu Y,Cheng M,Lu N et al.Construction of NK4 gene lentiviral vector and its expression in bone mesenchymal stem cells[J].Journal of Biomedical Engineering,2011,28(5):976-81.
[5]Doi R.Pancreatic tumor:progress in diagnosis and treatment.Topics:III.Pancreatic endocrine tumor;3.Multiple endocrine neoplasia[J].Nihon Naika Gakkai Zasshi,2012,101(1):116-9.
[6]Shi S,Yao W,Xu J,et al.Combinational therapy:new hope for pancreatic cancer?[J].Cancer Lett,2012,317(2):127-35.
[7]Chang D,Osman K,Connolly J,et al.Sustained expansion of NKT cells and antigen-specific T cells after injection of alphagalactosylceramide loaded mature dendritic cells in cancer patients[J].Exp Med,2005,201(9):1503-1517.
[8]Macho Fernandez E,Chang J,Fontaine J,et al.Activation of invariant Natural Killer T lymphocytes in response to theα-galactosylceramide analogue KRN7000 encapsulated in PLGA-based nanoparticles and microparticles[J].Int J Pharm,2012,423(1):45-54.
[9]Kuylenstierna C,Snyder-Cappione JE,Loo CP,et al.NK cells and CD1d-restricted NKT cells respond in different ways with divergent kinetics to IL-2 treatment in primary HIV-1 infection[J].Scand J Immunol,2011,73(2):141-146.
[10]Raghuraman G,Geng Y,Wang C R,IFN-b-Mediated Up-Regulation of CD1d in Bacteria-Inf-ected APCs[J].J Immunol,2006,177(11):7841-7848.
[11]师义,王昆华,刘为军,等.CD1d分子研究进展[J].广东医学,2012 33(11):1678-1680.
[12]Hix L M,Shi Y H,Brutkiewicz R R,et al.CD1d-expressing breast cancer cells modulate NKT cell-mediated antitumor immunity in a murine model of breast cancer metastasis[J].PLoS One,2011,6(6):e20702.
[13]Sun X Z,Liu G H,Wang Z Q,et al.Over-expression of VEGF165 in the adipose tissue-derived stem cells via the lentiviral vector[J].Chin Med J(Engl),2011,124(19):3093-7.
[14]Giry-Laterrière M,Verhoeyen E,Salmon P.Lentiviral vectors[J].Methods Mol Biol,2011,737:183-209.
[15]Xing H Y,Liu T,Gong Y P.Study of transfection of human embryonic stem cell with green fluorescent protein mediated by lentiviral vectors[J].Chinese Journal of Hematology,2011,32(5):349-51.