中华消化杂志
中華消化雜誌
중화소화잡지
Chinese Journal of Digestion
2013年
11期
750-755
,共6页
陈江%郭晓钟%胡志刚%李宏宇%王迪%赵佳钧
陳江%郭曉鐘%鬍誌剛%李宏宇%王迪%趙佳鈞
진강%곽효종%호지강%리굉우%왕적%조가균
树突细胞%黏蛋白类%端粒,末端转移酶%RNA,信使%胰腺肿瘤%T淋巴细胞,细胞毒性
樹突細胞%黏蛋白類%耑粒,末耑轉移酶%RNA,信使%胰腺腫瘤%T淋巴細胞,細胞毒性
수돌세포%점단백류%단립,말단전이매%RNA,신사%이선종류%T림파세포,세포독성
Dendritic cells%Mucins%Telomerase%RNA,messenger%Pancreatic neoplasms%T-lymphocytes,cytotoxic
目的 研究人胰腺癌黏蛋白4 mRNA与人端粒酶反转录酶(hTERT) mRNA联合转染树突状细胞(DC)诱导的抗肿瘤免疫反应,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6例HLA-A2+的胰腺癌患者外周血单个核细胞中分离、培养DC.体外转录和扩增黏蛋白4和hTERT mRNA,电穿孔法将两者分别及联合转染DC,培养48 h.采用实时定量PCR和Western印迹技术检测DC中黏蛋白4和hTERT的表达.用MTT法监测转染前后DC存活率；使用IFN-γ释放试验(ELISA法)检测黏蛋白4 mRNA和(或)hTERT mRNA转染后DC诱导的CTL的活化反应.采用51Cr标准细胞毒实验检测转染黏蛋白4 mRNA和(或)hTERT mRNA后DC诱导的CTL对体外胰腺癌细胞株MiaPaCa-2、Capan-2、AsPC-1和Panc-1细胞的杀伤作用.采用studentt检验进行统计学处理.结果 黏蛋白4 mRNA与hTERT mRNA联合转染后48 h DC中两者的相对表达量分别为30.09±5.24和12.87±3.36,低于其分别转染时的相对表达量(38.54土6.21和36.35+5.03,t=3.469、6.721,P均＜0.05).黏蛋白4 mRNA与hTERT mRNA联合转染后96h DC存活率降至52.17％,低于分别转染时DC的存活率(均为80％左右).黏蛋白4和hTERT mRNA联合转染DC诱导的特异性CTL 24 h IFN-γ释放量达(32.57±2.01) U/mL,高于分别转染时DC诱导的CTL IFN-γ释放水平[(23.06±4.74) U/mL和(16.82±3.67) U/mL,t=5.092和7.1 11,P均＜0.05].DC经黏蛋白4 mRNA与hTERT mRNA联合转染后,可有效诱导HLA-A2-黏蛋白4+/hTERT+特异性CTL免疫反应,而对HLA-A2-的胰腺癌细胞无显著杀伤作用.结论 黏蛋白4 mRNA与hTERT mRNA联合转染的DC较单胰腺癌抗原负载DC诱导出更加显著的CTL抗肿瘤免疫.
目的 研究人胰腺癌黏蛋白4 mRNA與人耑粒酶反轉錄酶(hTERT) mRNA聯閤轉染樹突狀細胞(DC)誘導的抗腫瘤免疫反應,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6例HLA-A2+的胰腺癌患者外週血單箇覈細胞中分離、培養DC.體外轉錄和擴增黏蛋白4和hTERT mRNA,電穿孔法將兩者分彆及聯閤轉染DC,培養48 h.採用實時定量PCR和Western印跡技術檢測DC中黏蛋白4和hTERT的錶達.用MTT法鑑測轉染前後DC存活率；使用IFN-γ釋放試驗(ELISA法)檢測黏蛋白4 mRNA和(或)hTERT mRNA轉染後DC誘導的CTL的活化反應.採用51Cr標準細胞毒實驗檢測轉染黏蛋白4 mRNA和(或)hTERT mRNA後DC誘導的CTL對體外胰腺癌細胞株MiaPaCa-2、Capan-2、AsPC-1和Panc-1細胞的殺傷作用.採用studentt檢驗進行統計學處理.結果 黏蛋白4 mRNA與hTERT mRNA聯閤轉染後48 h DC中兩者的相對錶達量分彆為30.09±5.24和12.87±3.36,低于其分彆轉染時的相對錶達量(38.54土6.21和36.35+5.03,t=3.469、6.721,P均＜0.05).黏蛋白4 mRNA與hTERT mRNA聯閤轉染後96h DC存活率降至52.17％,低于分彆轉染時DC的存活率(均為80％左右).黏蛋白4和hTERT mRNA聯閤轉染DC誘導的特異性CTL 24 h IFN-γ釋放量達(32.57±2.01) U/mL,高于分彆轉染時DC誘導的CTL IFN-γ釋放水平[(23.06±4.74) U/mL和(16.82±3.67) U/mL,t=5.092和7.1 11,P均＜0.05].DC經黏蛋白4 mRNA與hTERT mRNA聯閤轉染後,可有效誘導HLA-A2-黏蛋白4+/hTERT+特異性CTL免疫反應,而對HLA-A2-的胰腺癌細胞無顯著殺傷作用.結論 黏蛋白4 mRNA與hTERT mRNA聯閤轉染的DC較單胰腺癌抗原負載DC誘導齣更加顯著的CTL抗腫瘤免疫.
목적 연구인이선암점단백4 mRNA여인단립매반전록매(hTERT) mRNA연합전염수돌상세포(DC)유도적항종류면역반응,위구건부재다항원표위DC역묘치료이선암제공실험의거.방법 자6례HLA-A2+적이선암환자외주혈단개핵세포중분리、배양DC.체외전록화확증점단백4화hTERT mRNA,전천공법장량자분별급연합전염DC,배양48 h.채용실시정량PCR화Western인적기술검측DC중점단백4화hTERT적표체.용MTT법감측전염전후DC존활솔；사용IFN-γ석방시험(ELISA법)검측점단백4 mRNA화(혹)hTERT mRNA전염후DC유도적CTL적활화반응.채용51Cr표준세포독실험검측전염점단백4 mRNA화(혹)hTERT mRNA후DC유도적CTL대체외이선암세포주MiaPaCa-2、Capan-2、AsPC-1화Panc-1세포적살상작용.채용studentt검험진행통계학처리.결과 점단백4 mRNA여hTERT mRNA연합전염후48 h DC중량자적상대표체량분별위30.09±5.24화12.87±3.36,저우기분별전염시적상대표체량(38.54토6.21화36.35+5.03,t=3.469、6.721,P균＜0.05).점단백4 mRNA여hTERT mRNA연합전염후96h DC존활솔강지52.17％,저우분별전염시DC적존활솔(균위80％좌우).점단백4화hTERT mRNA연합전염DC유도적특이성CTL 24 h IFN-γ석방량체(32.57±2.01) U/mL,고우분별전염시DC유도적CTL IFN-γ석방수평[(23.06±4.74) U/mL화(16.82±3.67) U/mL,t=5.092화7.1 11,P균＜0.05].DC경점단백4 mRNA여hTERT mRNA연합전염후,가유효유도HLA-A2-점단백4+/hTERT+특이성CTL면역반응,이대HLA-A2-적이선암세포무현저살상작용.결론 점단백4 mRNA여hTERT mRNA연합전염적DC교단이선암항원부재DC유도출경가현저적CTL항종류면역.
Objective To investigate the anti-tumor immune response induced by human pancreatic cancer mucin 4 mRNA and human telomerase reverse transcriptase (hTERT) mRNA cotransfected dendritic cells (DC),and to provide the experimental evidences for the treatment of pancreatic cancer with multi-epitope loaded DC vaccine.Methods DC were isolated from peripheral blood mononuclear cells (PBMC) of six patients with HLA-A2+ pancreatic cancer and cultured.Mucin 4 mRNA and hTERT mRNA were transcripted and amplified in vitro,which were transfected into DC separately or in order by eleetroporation.DC were cultured for 48 hours.The expressions of mucin 4 and hTERT in DC were detected by quantitative real-time polymerase chain reaction (PCR) and Western blot.The survival rates of transfected DC were determined by methylthiazolyl tetrazolium (MTT) method.The cytotoxic T lymphocyte (CTL) activation induced by mucin 4 mRNA and hTERT mRNA transfected DC were evaluated by interferon (IFN)-γ release assays (enzyme linked immunosorbent assay) method.The cytotoxicity of CTL induced by mucin 4 mRNA and hTERT mRNA transfected DC in pancreatic cancer cell lines MiaPaCa-2,Capan-2,AsPC-1 and Pane-1 was measured by 51Cr standard cytotoxicity test.Student t test was performed for statistical analysis.Results After in order transfection of mucin 4 mRNA and hTERT mRNA for 48 h,the relative quantity of the expression of mucin 4 and hTERT in DC were 30.09±5.24 and 12.87±3.36,which were lower than the relative quantity of the expression in DC transfected separately (38.54±6.21 and 36.35±5.03,t=3.469,6.721,both P＜0.05).After transfected in order for 96 hours,the survival rate of DC decreased to 52.17％,which was lower than that of DC transfected separately (around 80％).The quantity of IFN-γ releasing of specific CTL induced by mucin 4 mRNA and hTERT mRNA cotransfected DC was (32.57±2.01) U/mL in 24 hours,which was higher than that of CTL induced by DC transfected with mucin 4 mRNA ((23.06±4.74) U/mL) or hTERT mRNA ((16.82±3.67) U/mL) separately (1=5.092,7.141,both P＜0.05).After co-transfected with mucin4 mRNA and hTERT mRNA,DC could effectivly induce HLA-A2+/mucin 4+/hTERT+ specific CTL immune responses,however there was no significant cytotoxicity in HLA-A2+ pancreatic cancer cells.Conclusion The induction of CTL anti-tumor immune response by DC co-transfected with mucin4 mRNA and hTERT mRNA is more significant compared with that by single antigen loaded DC.
