中华胰腺病杂志
中華胰腺病雜誌
중화이선병잡지
Chinese Journal of Pancreatology
2015年
5期
325-330
,共6页
卢学嘉%吕瑛%马超%刘文佳%谢丽娟%邹晓平
盧學嘉%呂瑛%馬超%劉文佳%謝麗娟%鄒曉平
로학가%려영%마초%류문가%사려연%추효평
胰腺肿瘤%树突细胞%肿瘤微环境%FOXP3
胰腺腫瘤%樹突細胞%腫瘤微環境%FOXP3
이선종류%수돌세포%종류미배경%FOXP3
Pancreatic neoplasms%Dendritic cells%Tumor microenvironment%FOXP3
目的 探讨胰腺癌细胞FOXP3表达对树突细胞( DCs )活化及其免疫功能的影响. 方法设计并合成靶向FOXP3的siRNA(siRNA-FOXP3)及阴性对照siRNA(siRNA-NC),转染胰腺癌PANC1细胞,ELISA法检测细胞培养上清IL-10和TGF-β1含量. 分别收集两组转染的胰腺癌细胞上清液,与粒细胞-单核细胞集落刺激因子( GM-CSF)及IL-4联合诱导DCs分化. 流式细胞仪检测经转染及未转染细胞培养上清液处理后DCs免疫表型CD86、CD80、HLA-DR等的变化,ELISA法检测上清中IL-12p70、IFN-γ含量. 将经上清液处理后的DCs与淋巴细胞共培养,CCK-8法检测各组DCs诱导的淋巴细胞增殖能力及活化的T细胞( CTLs)对胰腺癌细胞的杀伤性. 结果 与转染siRNA-NC的PANC1细胞比较,转染siRNA-FOXP3 的PANC1细胞IL-10、TGF-β1分泌量显著减少 [ (8.93 ±3.06)ng/L比(26.60 ± 5.57)ng/L,(2 544 ±78)ng/L比(2 856 ±92)ng/L],其细胞培养上清处理后DCs的CD86、HLA-DR阳性表达率显著增加[ (28.10 ±3.11)%比(13.90 ±0.42)%,(66.15 ±4.17)%比(43.15 ±3.32)% ],IL-12p70、IFN-γ分泌量显著增加[(52.75 ±7.89) ng/L比(26.14 ±4.50) ng/L,(898.43 ±88.82) ng/L比(412.76 ±24.68)ng/L],DCs 与淋巴细胞以1:5、1:10、1:20比例共培养后淋巴细胞的增殖显著加速[(95.27 ±3.80)%比(71.77 ±5.70)%,(78.97 ±5.73)%比(52.30 ±8.72)%,(57.60 ±4.36)%比(43.73 ±6.01)%],以1:20、1:40比例培养后活化的CTL对PANC1细胞的杀伤率显著提高[(28.44 ± 5.20)%比(8.82 ±2.29)%,(40.85 ±5.15)%比(17.38 ±4.86)%],两组间的差异均有统计学意义(P值均<0.05). 结论 胰腺癌细胞FOXP3表达显著抑制DCs的活化及其免疫功能.
目的 探討胰腺癌細胞FOXP3錶達對樹突細胞( DCs )活化及其免疫功能的影響. 方法設計併閤成靶嚮FOXP3的siRNA(siRNA-FOXP3)及陰性對照siRNA(siRNA-NC),轉染胰腺癌PANC1細胞,ELISA法檢測細胞培養上清IL-10和TGF-β1含量. 分彆收集兩組轉染的胰腺癌細胞上清液,與粒細胞-單覈細胞集落刺激因子( GM-CSF)及IL-4聯閤誘導DCs分化. 流式細胞儀檢測經轉染及未轉染細胞培養上清液處理後DCs免疫錶型CD86、CD80、HLA-DR等的變化,ELISA法檢測上清中IL-12p70、IFN-γ含量. 將經上清液處理後的DCs與淋巴細胞共培養,CCK-8法檢測各組DCs誘導的淋巴細胞增殖能力及活化的T細胞( CTLs)對胰腺癌細胞的殺傷性. 結果 與轉染siRNA-NC的PANC1細胞比較,轉染siRNA-FOXP3 的PANC1細胞IL-10、TGF-β1分泌量顯著減少 [ (8.93 ±3.06)ng/L比(26.60 ± 5.57)ng/L,(2 544 ±78)ng/L比(2 856 ±92)ng/L],其細胞培養上清處理後DCs的CD86、HLA-DR暘性錶達率顯著增加[ (28.10 ±3.11)%比(13.90 ±0.42)%,(66.15 ±4.17)%比(43.15 ±3.32)% ],IL-12p70、IFN-γ分泌量顯著增加[(52.75 ±7.89) ng/L比(26.14 ±4.50) ng/L,(898.43 ±88.82) ng/L比(412.76 ±24.68)ng/L],DCs 與淋巴細胞以1:5、1:10、1:20比例共培養後淋巴細胞的增殖顯著加速[(95.27 ±3.80)%比(71.77 ±5.70)%,(78.97 ±5.73)%比(52.30 ±8.72)%,(57.60 ±4.36)%比(43.73 ±6.01)%],以1:20、1:40比例培養後活化的CTL對PANC1細胞的殺傷率顯著提高[(28.44 ± 5.20)%比(8.82 ±2.29)%,(40.85 ±5.15)%比(17.38 ±4.86)%],兩組間的差異均有統計學意義(P值均<0.05). 結論 胰腺癌細胞FOXP3錶達顯著抑製DCs的活化及其免疫功能.
목적 탐토이선암세포FOXP3표체대수돌세포( DCs )활화급기면역공능적영향. 방법설계병합성파향FOXP3적siRNA(siRNA-FOXP3)급음성대조siRNA(siRNA-NC),전염이선암PANC1세포,ELISA법검측세포배양상청IL-10화TGF-β1함량. 분별수집량조전염적이선암세포상청액,여립세포-단핵세포집락자격인자( GM-CSF)급IL-4연합유도DCs분화. 류식세포의검측경전염급미전염세포배양상청액처리후DCs면역표형CD86、CD80、HLA-DR등적변화,ELISA법검측상청중IL-12p70、IFN-γ함량. 장경상청액처리후적DCs여림파세포공배양,CCK-8법검측각조DCs유도적림파세포증식능력급활화적T세포( CTLs)대이선암세포적살상성. 결과 여전염siRNA-NC적PANC1세포비교,전염siRNA-FOXP3 적PANC1세포IL-10、TGF-β1분비량현저감소 [ (8.93 ±3.06)ng/L비(26.60 ± 5.57)ng/L,(2 544 ±78)ng/L비(2 856 ±92)ng/L],기세포배양상청처리후DCs적CD86、HLA-DR양성표체솔현저증가[ (28.10 ±3.11)%비(13.90 ±0.42)%,(66.15 ±4.17)%비(43.15 ±3.32)% ],IL-12p70、IFN-γ분비량현저증가[(52.75 ±7.89) ng/L비(26.14 ±4.50) ng/L,(898.43 ±88.82) ng/L비(412.76 ±24.68)ng/L],DCs 여림파세포이1:5、1:10、1:20비례공배양후림파세포적증식현저가속[(95.27 ±3.80)%비(71.77 ±5.70)%,(78.97 ±5.73)%비(52.30 ±8.72)%,(57.60 ±4.36)%비(43.73 ±6.01)%],이1:20、1:40비례배양후활화적CTL대PANC1세포적살상솔현저제고[(28.44 ± 5.20)%비(8.82 ±2.29)%,(40.85 ±5.15)%비(17.38 ±4.86)%],량조간적차이균유통계학의의(P치균<0.05). 결론 이선암세포FOXP3표체현저억제DCs적활화급기면역공능.
Objective To investigate the influence of FOXP 3 expression in pancreatic carcinoma cells (PCCs) on the maturation and immunologic function of dendritic cells (DCs).Methods The siRNA sequences targeting FOXP3 gene (siRNA-FOXP3) and negative control siRNA (siRNA-NC) were specifically designed and transfected into PCCs , then the level of IL-10 and TGF-β1 of culture supernatant were detected by ELISA.The supernatants of pancreatic carcinoma cell transfected by FOXP 3-siRNA were collected , then it was mixed with GM-CSF and IL-4 to induce the differentiation of DCs .Flow cytometric analysis were used to measure the expression of surface markers CD 86 , CD80 , HLA DR on DCs which were treated with supernatants . The levels of IL-12p70, IFN-γin supernatants were detected by ELISA .The DCs were co-cultured with T lymphocytes, and then the lymphocytes proliferation and cytoxicity were analyzed by CCK -8 assays.Results Compared with PANC1 with siRNA-NC transfection, PANC1 with siRNA-FOXP3 transfection had a decreased expression of IL-10, TGF-β1 [(8.93 ±3.06)ng/L vs (26.60 ±5.57)ng/L;(2 544 ±78)ng/L vs (2 856 ± 92)ng/L], the positive expression rate of CD86, HLA DR in DCs cultured in the medium containing the supernatants of the pancreatic carcinoma cell transfected by siRNA-FOXP3 was significantly increased [(28.10 ±3.11)%vs (13.90 ±0.42)%;(66.15 ±4.17)%vs (43.15 ±3.32)%], the expression of IL-12p70, IFN-γwas significantly increased [(52.75 ±7.89)ng/L vs (26.14 ±4.50)ng/L, (898.43 ±88.82) ng/L vs (412.76 ±24.68) ng/L], after co-culture with lymphocytes at ratios of 1:5, 1:10, 1:20, the proliferation was significantly increased [(95.27 ±3.80)% vs (71.77 ±5.70)%, (78.97 ±5.73)% vs (52.30 ±8.72)%, (57.60 ±4.36)% vs (43.73 ±6.01)%], and the cytoxicity of CTL to PANC1 cells with 1:20, 1:40 co-culture was significantly increased [(28.44 ±5.20)% vs (8.82 ±2.29)%, (40.85 ± 5.15)% vs (17.38 ±4.86)%], and the difference between the two groups was statistically significant (P<0.05).Conclusions FOXP3 expression in PCCs can inhibit the maturation and immunologic function of DCs.
