中华肿瘤杂志
中華腫瘤雜誌
중화종류잡지
CHINESE JOURNAL OF ONCOLOGY
2012年
5期
336-340
,共5页
尤红煜%连伟光%张焕铃%王俊霞%张开霞%宋淑霞
尤紅煜%連偉光%張煥鈴%王俊霞%張開霞%宋淑霞
우홍욱%련위광%장환령%왕준하%장개하%송숙하
黑色素瘤,实验性%顺铂%树突状细胞,疫苗%小鼠%调节性T细胞%肿瘤浸润淋巴细胞%细胞毒性淋巴细胞
黑色素瘤,實驗性%順鉑%樹突狀細胞,疫苗%小鼠%調節性T細胞%腫瘤浸潤淋巴細胞%細胞毒性淋巴細胞
흑색소류,실험성%순박%수돌상세포,역묘%소서%조절성T세포%종류침윤림파세포%세포독성림파세포
Melanoma,experimental%Cisplatin%Vaccines,dendritic cells%Mice%Regulatory T cells%Tumor infiltrating lymphocytes%Cytotoxic lymphocytes
目的 探讨顺铂联合树突状细胞(DC)疫苗对荷瘤小鼠抗肿瘤作用的机制.方法 用终浓度20μg/ml顺铂诱导体外培养的小鼠黑色素瘤B16细胞凋亡,提取凋亡细胞总蛋白.Westernblot法检测高迁移率蛋白B1( HMGB1)、热休克蛋白70 (Hsp70)和转化生长因子β(TGF-β)蛋白表达,流式细胞仪分析DCs表面主要组织相容性复合体Ⅱ类(MHCⅡ)、细胞间黏附分子1(ICAM-1)和CD86的表达.制备荷瘤小鼠模型,腹腔注射顺铂(100μg/只),瘤内注射DC疫苗(3×106个/只),28 d后处死荷瘤小鼠,分离肿瘤组织并称重.采用不连续梯度离心法分离肿瘤浸润淋巴细胞,流式细胞仪检测调节性T细胞(T-reg)和CD8+T细胞的分布,采用微量细胞毒方法检测细胞毒性T淋巴细胞(CTL)活性.结果 顺铂可明显提高B16细胞HMGB1的表达水平.经顺铂诱导的B16凋亡细胞总蛋白负载DC细胞后,其MHCⅡ、CD86+和ICAM-1的表达率分别为(47.5±8.8)％、(36.2±9.2)％和(35.5±8.3)％.在肿瘤移植的第28天,对照组瘤重为(2.1±0.6)g,顺铂组和顺铂联合DC疫苗组瘤重分别为(0.3±0.2)g和(0.5±0.2)g,顺铂组和顺铂联合DC疫苗组瘤重明显小于对照组(P＜0.01).顺铂联合DC疫苗不仅提高了肿瘤组织中CD8+ T/CD4+ Foxp3+T细胞比值,还增强了 CTL活性.在效靶比为20∶1、10∶1和5∶1时,顺铂联合DC疫苗组荷瘤小鼠的CTL活性分别为(25.0±5.0)％、(22.0±6.0)％和(14.0±4.0)％,显著高于对照组的(8.2±3.6)％、(6.7±1.8)％和(3.6±1.9)％(均P＜0.01).结论 顺铂联合DC疫苗可下调肿瘤微环境的T-reg细胞,提高CTL活性协同发挥抗肿瘤作用.
目的 探討順鉑聯閤樹突狀細胞(DC)疫苗對荷瘤小鼠抗腫瘤作用的機製.方法 用終濃度20μg/ml順鉑誘導體外培養的小鼠黑色素瘤B16細胞凋亡,提取凋亡細胞總蛋白.Westernblot法檢測高遷移率蛋白B1( HMGB1)、熱休剋蛋白70 (Hsp70)和轉化生長因子β(TGF-β)蛋白錶達,流式細胞儀分析DCs錶麵主要組織相容性複閤體Ⅱ類(MHCⅡ)、細胞間黏附分子1(ICAM-1)和CD86的錶達.製備荷瘤小鼠模型,腹腔註射順鉑(100μg/隻),瘤內註射DC疫苗(3×106箇/隻),28 d後處死荷瘤小鼠,分離腫瘤組織併稱重.採用不連續梯度離心法分離腫瘤浸潤淋巴細胞,流式細胞儀檢測調節性T細胞(T-reg)和CD8+T細胞的分佈,採用微量細胞毒方法檢測細胞毒性T淋巴細胞(CTL)活性.結果 順鉑可明顯提高B16細胞HMGB1的錶達水平.經順鉑誘導的B16凋亡細胞總蛋白負載DC細胞後,其MHCⅡ、CD86+和ICAM-1的錶達率分彆為(47.5±8.8)％、(36.2±9.2)％和(35.5±8.3)％.在腫瘤移植的第28天,對照組瘤重為(2.1±0.6)g,順鉑組和順鉑聯閤DC疫苗組瘤重分彆為(0.3±0.2)g和(0.5±0.2)g,順鉑組和順鉑聯閤DC疫苗組瘤重明顯小于對照組(P＜0.01).順鉑聯閤DC疫苗不僅提高瞭腫瘤組織中CD8+ T/CD4+ Foxp3+T細胞比值,還增彊瞭 CTL活性.在效靶比為20∶1、10∶1和5∶1時,順鉑聯閤DC疫苗組荷瘤小鼠的CTL活性分彆為(25.0±5.0)％、(22.0±6.0)％和(14.0±4.0)％,顯著高于對照組的(8.2±3.6)％、(6.7±1.8)％和(3.6±1.9)％(均P＜0.01).結論 順鉑聯閤DC疫苗可下調腫瘤微環境的T-reg細胞,提高CTL活性協同髮揮抗腫瘤作用.
목적 탐토순박연합수돌상세포(DC)역묘대하류소서항종류작용적궤제.방법 용종농도20μg/ml순박유도체외배양적소서흑색소류B16세포조망,제취조망세포총단백.Westernblot법검측고천이솔단백B1( HMGB1)、열휴극단백70 (Hsp70)화전화생장인자β(TGF-β)단백표체,류식세포의분석DCs표면주요조직상용성복합체Ⅱ류(MHCⅡ)、세포간점부분자1(ICAM-1)화CD86적표체.제비하류소서모형,복강주사순박(100μg/지),류내주사DC역묘(3×106개/지),28 d후처사하류소서,분리종류조직병칭중.채용불련속제도리심법분리종류침윤림파세포,류식세포의검측조절성T세포(T-reg)화CD8+T세포적분포,채용미량세포독방법검측세포독성T림파세포(CTL)활성.결과 순박가명현제고B16세포HMGB1적표체수평.경순박유도적B16조망세포총단백부재DC세포후,기MHCⅡ、CD86+화ICAM-1적표체솔분별위(47.5±8.8)％、(36.2±9.2)％화(35.5±8.3)％.재종류이식적제28천,대조조류중위(2.1±0.6)g,순박조화순박연합DC역묘조류중분별위(0.3±0.2)g화(0.5±0.2)g,순박조화순박연합DC역묘조류중명현소우대조조(P＜0.01).순박연합DC역묘불부제고료종류조직중CD8+ T/CD4+ Foxp3+T세포비치,환증강료 CTL활성.재효파비위20∶1、10∶1화5∶1시,순박연합DC역묘조하류소서적CTL활성분별위(25.0±5.0)％、(22.0±6.0)％화(14.0±4.0)％,현저고우대조조적(8.2±3.6)％、(6.7±1.8)％화(3.6±1.9)％(균P＜0.01).결론 순박연합DC역묘가하조종류미배경적T-reg세포,제고CTL활성협동발휘항종류작용.
Objective To explore the anti-tumor mechanism of the combination of cisplatin with DC vaccine in tumor-bearing mice.Methods B16 melanoma cells were treated with cisplatin at the final concentration of 20 μg/ml in vitro for 24 h.The expression of HMGB1,Hsp70 and TGF-β were detected by Western blot.B16 turmor-bearing mouse models were generated.The therapeutic effect of the combination of cisplatin (100 μg/mouse i.p.,for sequential 3 days) and intratumoral injection of DC cells (3 × 106/mouse,twice with a 7-day interval) in the tumor-bearing mouse models was evaluated.Expression of MHC Ⅱ,ICAM-1 and CD86 was analyzed by flow cytometry.The mice were sacrificed at 28 days after tumor cell inoculation.The tumors were removed and weighed,and tissue samples were taken for pathological examination.Tumor infiltrating lymphocytes (TIL) were isolated by discontinuous gradient centrifugation.The distribution of T-reg and CD8+ T cells in the TIL was analyzed by flow cytometry,and the ratio of CD8 +T/T-reg was determined.The activity of cytotoxic lymphocytes (CTL) was determined by microcytotoxicity assay.Results Cisplatin enhanced both the B16 cell apoptosis and HMGB1 expression.After loading with cisplatin-treated cell lysate,the expression of MHC Ⅱ,ICAM-1 and CD86 on DC cells were (47.5 ±8.8) ％,(35.5 ± 8.3 ) ％ and (36.2 ± 9.2) ％,respectively.At 28 days after tumor cell inoculation,the tumor weight of the control group was (2.1 ± 0.6) g,that of the cisplatin group was (0.3 ± 0.2) g and that of cisplatin + DC vaccine group was (0.5 ± 0.2) g,showing a significant inhibition of tumor growth ( P ＜0.01 ).Furthermore,the CD8 + T/T-reg ratio and CTL activity in TIL were also significantly enhanced in the tumor-bearing mice treated with cisplatin + DC vaccine.When the effector-to-target ratio was 20∶ 1,10∶1 and 5∶1,the CTL activity in the cisplatin + DC vaccine treated mice was (25.0 ± 5.0)％,(22.0±6.0) ％ and ( 14.0 ± 4.0 ) ％,respectively,significantly higher than ( 8.2 ± 3.6 ) ％,( 6.7 ± 1.8 ) ％ and (3.6±1.9)％,respectively,in the control group (all P＜0.01).Conclusion Cisplatin promotes the anti-tumor effect of DC vaccine by down-regulating T-reg cells and enhancing the CTL activity in tumors.
