CAJ | 학술논문

目的：研究人胰腺癌MUC4与Survivin mRNA联合转染树突细胞（DC）诱导的特异性抗肿瘤免疫反应，为构建负载多抗原表位DC疫苗治疗胰腺癌提供实验依据。方法自胰腺癌患者外周血单核细胞中分离、培养DCs。使用体外转录和胰腺癌PCR技术扩增MUC4和Survivin mRNA后用电穿孔法将其联合转染DC。采用Western blot技术检测DCs中MUC4和Survivin的表达。用四甲基偶氮唑盐（MTT）法检测转染前后DCs存活率变化；使用IFN-γ酶联免疫法检测MUC4 mRNA与Survivin mRNA联合转染后DC诱导的细胞毒性T淋巴细胞（CTL）的活化反应。采用51Cr标准细胞毒实验检测转染MUC4和Survivin mRNA后DCs诱导的特异性CTL对体外胰腺癌细胞的杀伤作用。结果 MUC4与Survivin mRNA联合转染后72 h DCs中两者的相对表达量低于其分别转染。顺序转染后96 h DCs存活率降至50.2%，低于MUC4 mRNA与Survivin mRNA分别转染时DC 80%的存活率（P<0.05）。MUC4和Survivin mRNA联合转染DC诱导的特异性CTL 24 h IFN-γ释放量达（33.84±3.51）U/mL，高于MUC4与Survivin mRNA分别转染DC诱导的CTL IFN-γ释放水平[（21.87±4.12）U/mL和（16.61±2.09）U/mL，P<0.05]。 DCs经MUC4 mRNA与Survivin mRNA联合转染后，可有效诱导HLA-A2+/MUC4+/Survivin+特异性CTL免疫反应，对体外培养的胰腺癌细胞具有显著的杀伤作用。结论 MUC4与Sur-vivin mRNA联合转染的DCs可较单胰腺癌相关抗原负载DCs诱导出更加显著的特异性CTL抗肿瘤免疫。amount of (21.87±4.12)U/ml by MUC4 or (16.61±2.09)U/ml by Survivin mRNA individually (P<0.05). DCs co-transfection with MUC4 and Survivin mRNA could effectively induce HLA-A2+/MUC4+/Survivin+specific CTL immune responses against pancreatic cancer cells in vitro. Conclusion The induction of CTLs by DCs co-transfected with human pancreatic cancer MUC4 and Survivin mRNA could produce more powerful specific anti-tumor immunity than single antigen loaded DCs. 目的：研究人胰腺癌MUC4與Survivin mRNA聯閤轉染樹突細胞（DC）誘導的特異性抗腫瘤免疫反應，為構建負載多抗原錶位DC疫苗治療胰腺癌提供實驗依據。方法自胰腺癌患者外週血單覈細胞中分離、培養DCs。使用體外轉錄和胰腺癌PCR技術擴增MUC4和Survivin mRNA後用電穿孔法將其聯閤轉染DC。採用Western blot技術檢測DCs中MUC4和Survivin的錶達。用四甲基偶氮唑鹽（MTT）法檢測轉染前後DCs存活率變化；使用IFN-γ酶聯免疫法檢測MUC4 mRNA與Survivin mRNA聯閤轉染後DC誘導的細胞毒性T淋巴細胞（CTL）的活化反應。採用51Cr標準細胞毒實驗檢測轉染MUC4和Survivin mRNA後DCs誘導的特異性CTL對體外胰腺癌細胞的殺傷作用。結果 MUC4與Survivin mRNA聯閤轉染後72 h DCs中兩者的相對錶達量低于其分彆轉染。順序轉染後96 h DCs存活率降至50.2%，低于MUC4 mRNA與Survivin mRNA分彆轉染時DC 80%的存活率（P<0.05）。MUC4和Survivin mRNA聯閤轉染DC誘導的特異性CTL 24 h IFN-γ釋放量達（33.84±3.51）U/mL，高于MUC4與Survivin mRNA分彆轉染DC誘導的CTL IFN-γ釋放水平[（21.87±4.12）U/mL和（16.61±2.09）U/mL，P<0.05]。 DCs經MUC4 mRNA與Survivin mRNA聯閤轉染後，可有效誘導HLA-A2+/MUC4+/Survivin+特異性CTL免疫反應，對體外培養的胰腺癌細胞具有顯著的殺傷作用。結論 MUC4與Sur-vivin mRNA聯閤轉染的DCs可較單胰腺癌相關抗原負載DCs誘導齣更加顯著的特異性CTL抗腫瘤免疫。amount of (21.87±4.12)U/ml by MUC4 or (16.61±2.09)U/ml by Survivin mRNA individually (P<0.05). DCs co-transfection with MUC4 and Survivin mRNA could effectively induce HLA-A2+/MUC4+/Survivin+specific CTL immune responses against pancreatic cancer cells in vitro. Conclusion The induction of CTLs by DCs co-transfected with human pancreatic cancer MUC4 and Survivin mRNA could produce more powerful specific anti-tumor immunity than single antigen loaded DCs.
목적：연구인이선암MUC4여Survivin mRNA연합전염수돌세포（DC）유도적특이성항종류면역반응，위구건부재다항원표위DC역묘치료이선암제공실험의거。방법자이선암환자외주혈단핵세포중분리、배양DCs。사용체외전록화이선암PCR기술확증MUC4화Survivin mRNA후용전천공법장기연합전염DC。채용Western blot기술검측DCs중MUC4화Survivin적표체。용사갑기우담서염（MTT）법검측전염전후DCs존활솔변화；사용IFN-γ매련면역법검측MUC4 mRNA여Survivin mRNA연합전염후DC유도적세포독성T림파세포（CTL）적활화반응。채용51Cr표준세포독실험검측전염MUC4화Survivin mRNA후DCs유도적특이성CTL대체외이선암세포적살상작용。결과 MUC4여Survivin mRNA연합전염후72 h DCs중량자적상대표체량저우기분별전염。순서전염후96 h DCs존활솔강지50.2%，저우MUC4 mRNA여Survivin mRNA분별전염시DC 80%적존활솔（P<0.05）。MUC4화Survivin mRNA연합전염DC유도적특이성CTL 24 h IFN-γ석방량체（33.84±3.51）U/mL，고우MUC4여Survivin mRNA분별전염DC유도적CTL IFN-γ석방수평[（21.87±4.12）U/mL화（16.61±2.09）U/mL，P<0.05]。 DCs경MUC4 mRNA여Survivin mRNA연합전염후，가유효유도HLA-A2+/MUC4+/Survivin+특이성CTL면역반응，대체외배양적이선암세포구유현저적살상작용。결론 MUC4여Sur-vivin mRNA연합전염적DCs가교단이선암상관항원부재DCs유도출경가현저적특이성CTL항종류면역。amount of (21.87±4.12)U/ml by MUC4 or (16.61±2.09)U/ml by Survivin mRNA individually (P<0.05). DCs co-transfection with MUC4 and Survivin mRNA could effectively induce HLA-A2+/MUC4+/Survivin+specific CTL immune responses against pancreatic cancer cells in vitro. Conclusion The induction of CTLs by DCs co-transfected with human pancreatic cancer MUC4 and Survivin mRNA could produce more powerful specific anti-tumor immunity than single antigen loaded DCs.
Objective To investigate the induction of specific anti-tumor immune response induced by MUC4 and Survivin mRNA co-transfected dendritic cells (DCs) to provide the experimental evidences for the treatment of human pancreatic cancer with multi-epitope loaded DC vaccine. Methods DCs were isolated and cultured from peripheral blood mononuclear cells (PBMCs). After being transcripted and amplified, MUC4 and Survivin mRNA were co-transfected into DCs by electroporation. The expression of MUC4 and Survivin in DCs were detected by Western blot. The survival rate of transfected DCs were determined by MTT method. The induction of specific CTL activation by MUC4 and Survivin mRNA co-transfected DCs were evaluated through testing released IFN-γ by ELISA method. The induction of specific cytotoxic T lymphocyte (CTL) re-sponse by MUC4 and Survivin mRNA co-transfected DCs were measured by 51Cr standard cytotoxicity test. Results After MUC4 and Survivin mRNA co-transfection for 72 hours, the expression amount of MUC4 and Survivin were lower than the expression amount of MUC4 or Survivin individually. Compared with the MUC4 or Survivin mRNA individual transfected DCs, the IFN-γreleased in 24 hours by CTLs stimulated with MUC4 and Survivin mRNA co-transfection DCs were (33.84 ± 3.51)U/ml which was significantly higher than the amount of (21.87 ± 4.12)U/ml by MUC4 or (16.61 ± 2.09)U/ml by Survivin mRNA individually (P < 0.05). DCs co-transfection with MUC4 and Survivin mRNA could effectively induce HLA-A2 +/MUC4+/Survivin + specific CTL immune responses against pancreatic cancer cells in vitro. Conclusion The induction of CTLs by DCs co-transfected with human pancreatic cancer MUC4 and Survivin mRNA could produce more powerful specific anti-tumor immunity than single antigen loaded DCs.