中华胰腺病杂志
中華胰腺病雜誌
중화이선병잡지
CHINESE JOURNAL OF PANCREATOLOGY
2014年
6期
380-384
,共5页
陈江%牟为民%李宏宇%王迪%郭晓钟
陳江%牟為民%李宏宇%王迪%郭曉鐘
진강%모위민%리굉우%왕적%곽효종
树突细胞%RNA转染%胰腺肿瘤%T淋巴细胞,细胞毒性
樹突細胞%RNA轉染%胰腺腫瘤%T淋巴細胞,細胞毒性
수돌세포%RNA전염%이선종류%T림파세포,세포독성
Dendritic cells%RNA transfection%Pancreatic neoplasms%T-lymphocytes,cytotoxic
目的 观察人胰腺癌Capan-2细胞总RNA转染的树突细胞(DCs)所诱导的抗肿瘤免疫反应.方法 从6例胰腺癌患者外周血单核细胞中分离、培养DCs.使用电穿孔法将Capan-2细胞总RNA及MUC4 mRNA分别转染DCs.应用四甲基偶氮唑蓝(MTT)法检测转染后DCs的存活率.采用蛋白质印迹法检测DCs中MUC4 mRNA的表达.使用IFN-γ酶联免疫法检测DCs诱导的细胞毒T淋巴细胞(CTLs)的活化反应.采用51Cr标准细胞毒实验检测DCs诱导的抗原特异性CTLs对体外胰腺癌细胞的杀伤效应.结果 Capan-2细胞总RNA转染的DCs(DC-Capan-2-total RNA)的存活率呈时间依赖性下降,转染后96 h的存活率降低至60.8％,而转染MUC4 mRNA的DCs(DC-MUC4 mRNA)的存活率稳定在80.0％左右,两转染组DCs的差异具有统计学意义(P＜0.05).DC-Capan-2-total RNA的MUC4蛋白表达量亦显著低于DC-MUC4 mRNA(P＜0.05).DC-Capan-2-total RNA诱导的CTLs 24 h IFN-γ释放量为(89.34±3.85) U/ml,DC-MUC4 mRNA为(21.77±2.14) U/ml,两转染组的差异有统计学意义(P＜0.05).DC-Capan-2-total RNA诱导的特异性CTLs能够有效识别和杀伤HLA-A2 +/MUC4+的Capan-2细胞及HLA-A2 +/MUC4-的PANC1细胞,而不能有效识别和杀伤HLA-A2-/MUC4-的MiaPaCa-2细胞和HLA-A2-/MUC4+的AsPC-1细胞.结论 胰腺癌细胞总RNA转染的DCs较单个胰腺癌相关抗原转染的DCs能诱导出更加显著的CTLs抗肿瘤免疫反应,但受到MHC Ⅰ类抗原递呈的限制.
目的 觀察人胰腺癌Capan-2細胞總RNA轉染的樹突細胞(DCs)所誘導的抗腫瘤免疫反應.方法 從6例胰腺癌患者外週血單覈細胞中分離、培養DCs.使用電穿孔法將Capan-2細胞總RNA及MUC4 mRNA分彆轉染DCs.應用四甲基偶氮唑藍(MTT)法檢測轉染後DCs的存活率.採用蛋白質印跡法檢測DCs中MUC4 mRNA的錶達.使用IFN-γ酶聯免疫法檢測DCs誘導的細胞毒T淋巴細胞(CTLs)的活化反應.採用51Cr標準細胞毒實驗檢測DCs誘導的抗原特異性CTLs對體外胰腺癌細胞的殺傷效應.結果 Capan-2細胞總RNA轉染的DCs(DC-Capan-2-total RNA)的存活率呈時間依賴性下降,轉染後96 h的存活率降低至60.8％,而轉染MUC4 mRNA的DCs(DC-MUC4 mRNA)的存活率穩定在80.0％左右,兩轉染組DCs的差異具有統計學意義(P＜0.05).DC-Capan-2-total RNA的MUC4蛋白錶達量亦顯著低于DC-MUC4 mRNA(P＜0.05).DC-Capan-2-total RNA誘導的CTLs 24 h IFN-γ釋放量為(89.34±3.85) U/ml,DC-MUC4 mRNA為(21.77±2.14) U/ml,兩轉染組的差異有統計學意義(P＜0.05).DC-Capan-2-total RNA誘導的特異性CTLs能夠有效識彆和殺傷HLA-A2 +/MUC4+的Capan-2細胞及HLA-A2 +/MUC4-的PANC1細胞,而不能有效識彆和殺傷HLA-A2-/MUC4-的MiaPaCa-2細胞和HLA-A2-/MUC4+的AsPC-1細胞.結論 胰腺癌細胞總RNA轉染的DCs較單箇胰腺癌相關抗原轉染的DCs能誘導齣更加顯著的CTLs抗腫瘤免疫反應,但受到MHC Ⅰ類抗原遞呈的限製.
목적 관찰인이선암Capan-2세포총RNA전염적수돌세포(DCs)소유도적항종류면역반응.방법 종6례이선암환자외주혈단핵세포중분리、배양DCs.사용전천공법장Capan-2세포총RNA급MUC4 mRNA분별전염DCs.응용사갑기우담서람(MTT)법검측전염후DCs적존활솔.채용단백질인적법검측DCs중MUC4 mRNA적표체.사용IFN-γ매련면역법검측DCs유도적세포독T림파세포(CTLs)적활화반응.채용51Cr표준세포독실험검측DCs유도적항원특이성CTLs대체외이선암세포적살상효응.결과 Capan-2세포총RNA전염적DCs(DC-Capan-2-total RNA)적존활솔정시간의뢰성하강,전염후96 h적존활솔강저지60.8％,이전염MUC4 mRNA적DCs(DC-MUC4 mRNA)적존활솔은정재80.0％좌우,량전염조DCs적차이구유통계학의의(P＜0.05).DC-Capan-2-total RNA적MUC4단백표체량역현저저우DC-MUC4 mRNA(P＜0.05).DC-Capan-2-total RNA유도적CTLs 24 h IFN-γ석방량위(89.34±3.85) U/ml,DC-MUC4 mRNA위(21.77±2.14) U/ml,량전염조적차이유통계학의의(P＜0.05).DC-Capan-2-total RNA유도적특이성CTLs능구유효식별화살상HLA-A2 +/MUC4+적Capan-2세포급HLA-A2 +/MUC4-적PANC1세포,이불능유효식별화살상HLA-A2-/MUC4-적MiaPaCa-2세포화HLA-A2-/MUC4+적AsPC-1세포.결론 이선암세포총RNA전염적DCs교단개이선암상관항원전염적DCs능유도출경가현저적CTLs항종류면역반응,단수도MHC Ⅰ류항원체정적한제.
Objective To investigate the specific anti-tumor immune response induced by dendritic cells (DCs) transfected with total RNA of human pancreatic cancer Capan-2 cells.Methods DCs were isolated and cultured from peripheral blood mononuclear cells (PBMCs) derived from six patients with pancreatic cancer.Total RNA of Capan-2 cells and MUC4 mRNA were transfected into DCs by electroporation.The survival rate of transfected DCs was determined by MTT method and the expression of MUC4 mRNA in DCs was detected by Western blotting.The activity of cytotoxic T lymphocyte cells (CTLs) induced by DCs transfected with total RNA of Capan-2 cells were evaluated by IFN-γ ELISA and the induction of specific CTL response to the killing effect on pancreatic cancer cell in vitro were measured by 51 Cr standard cytotoxicity test.Results The survival rate of DCs transfected with total RNA of Capan-2 cells (DC-Capan-2-total RNA) showed a decrease in a time dependent manner and the survival rate was reduced to 60.81％ after transfection for 96 h.The survival rate of MUC4 mRNA transfection DCs (DC-MUC4 mRNA) was stable at around 80％.The difference of DCs surviral rate between the two groups was statistically significant (P ＜ 0.05).The amount of MUC4 protein expression of DC-Capan-2-total RNA was significantly lower than that of DC-MUC4 mRNA (P ＜0.05).The quantity of CTL IFN-γ release induced by DC-Capan-2-total RNA was (89.34 ± 3.85)U/mL and the quantity of DC-MUC4 mRNA induced CTL IFN-γ release was (21.77 ± 21.77)U/ml There was statistically significant between the two groups (P ＜0.05).In addition,the specific CTLs induced by DC-Capan-2-total RNA could effectively identify and kill the HLA-A2+/MUC4+ Capan-2 and the HLA-A2+/MUC4-PANC 1 cells,and could not effectively identify and kill the HLA-A2 /MUC4-MiaPaCa-2 cells and the HLA-A2-/MUC4 + AsPC-1 cells.Conclusions A more pronounced CTL anti-tumor immune response can be induced by DCs transfected with total RNA of Capan-2 cells compared with a single tumor associated antigen,but it is limited by MHC class Ⅰ antigen presented.
