中国骨与关节杂志
中國骨與關節雜誌
중국골여관절잡지
Chinese Journal of Bone and Joint
2014年
10期
786-790
,共5页
彭伟%黄迅悟%赵伟鹏%赵铭%杨大志
彭偉%黃迅悟%趙偉鵬%趙銘%楊大誌
팽위%황신오%조위붕%조명%양대지
肽类%药物筛选试验,抗肿瘤%抗肿瘤药%限制性表位肽%尤文肉瘤
肽類%藥物篩選試驗,抗腫瘤%抗腫瘤藥%限製性錶位肽%尤文肉瘤
태류%약물사선시험,항종류%항종류약%한제성표위태%우문육류
Peptides%Drug screening assays,antitumor%Antineoplastic agents%Restrictive epitope peptide%Ewing's sarcoma
目的比较不同EWS/FLI-1蛋白HLA-A/0201限制性表位肽对尤文肉瘤树突状细胞(dendriticcell，DC)疫苗的抗肿瘤作用。方法综合运用BIMAS、SYFPEITHI软件筛选与人白细胞抗原HLA-A/0201结合力强的EWS/FLI-1蛋白9肽表位，并合成其表位肽。采用4 h标准51Cr释放实验检测刺激细胞毒性T淋巴细胞( cytotoxic T lymphocytes，CTLs )对肿瘤细胞的杀伤效应。应用酶联免疫斑点法( enzyme linked immunospot assay，ELISPOT )检测表位肽对DC疫苗刺激的效应细胞γ-干扰素( interferon，IFN-γ)的释放。在此基础上，进行免疫治疗的动物实验，比较其体内抗肿瘤作用。结果筛选出EWS306、EWS289及EWS 401与HLA-A/0201具较强的结合性，其合成出的表位肽分别为：QLWQFLLEL ( EWS 306)、ILGPTSSRL ( EWS 289)和SMYKYPSDI ( EWS 401)。3组表位肽均有较强的杀伤力，当效-靶比为50∶1时，杀伤率EWS 306组(20.2±1.8)%、EWS 289组(12.6±0.3)%、EWS 401组(11.9±0.2)%，而对照组为(6.7±0.1)%；当效-靶比为100∶1时，表位肽的三组杀伤作用明显提升，杀伤率EWS 306组最高为(51.2±3.7)%、EWS 289组(24.6±2.1)%、EWS 401组(17.8±0.9)%，对照组为(7.2±0.2)%。EWS 306组同其它组比较，差异有统计学( P＜0.05)。EWS 306组分泌的IFN-γ为(118.3±3.6)个点明显高于EWS401组(35.1±1.0)个和EWS 289组(34.2±0.9)个，对照组为(5.0±0.1)个( P＜0.05)。EWS 306组肿瘤的体积(978.9±28.2) mm3明显小于阴性对照组(1992.9±16.1) mm3和空白对照组(2001.9±12.3) mm3( P＜0.05)。接种后第35天，EWS 306组存活率为80%大于空白对照组为0%、阴性对照组为10%( P＜0.05)；接种后第40天，空白对照组降为0%， EWS306组为80%明显大于和2个对照组(P＜0.05)。结论 EWS/FLI-1蛋白表位肽中EWS306较EWS289和EWS 401对DC的抗肿瘤作用更强，能够有效激活CTLs对肿瘤细胞的杀伤效应，为尤文肉瘤的免疫治疗提供了新的方向。
目的比較不同EWS/FLI-1蛋白HLA-A/0201限製性錶位肽對尤文肉瘤樹突狀細胞(dendriticcell，DC)疫苗的抗腫瘤作用。方法綜閤運用BIMAS、SYFPEITHI軟件篩選與人白細胞抗原HLA-A/0201結閤力彊的EWS/FLI-1蛋白9肽錶位，併閤成其錶位肽。採用4 h標準51Cr釋放實驗檢測刺激細胞毒性T淋巴細胞( cytotoxic T lymphocytes，CTLs )對腫瘤細胞的殺傷效應。應用酶聯免疫斑點法( enzyme linked immunospot assay，ELISPOT )檢測錶位肽對DC疫苗刺激的效應細胞γ-榦擾素( interferon，IFN-γ)的釋放。在此基礎上，進行免疫治療的動物實驗，比較其體內抗腫瘤作用。結果篩選齣EWS306、EWS289及EWS 401與HLA-A/0201具較彊的結閤性，其閤成齣的錶位肽分彆為：QLWQFLLEL ( EWS 306)、ILGPTSSRL ( EWS 289)和SMYKYPSDI ( EWS 401)。3組錶位肽均有較彊的殺傷力，噹效-靶比為50∶1時，殺傷率EWS 306組(20.2±1.8)%、EWS 289組(12.6±0.3)%、EWS 401組(11.9±0.2)%，而對照組為(6.7±0.1)%；噹效-靶比為100∶1時，錶位肽的三組殺傷作用明顯提升，殺傷率EWS 306組最高為(51.2±3.7)%、EWS 289組(24.6±2.1)%、EWS 401組(17.8±0.9)%，對照組為(7.2±0.2)%。EWS 306組同其它組比較，差異有統計學( P＜0.05)。EWS 306組分泌的IFN-γ為(118.3±3.6)箇點明顯高于EWS401組(35.1±1.0)箇和EWS 289組(34.2±0.9)箇，對照組為(5.0±0.1)箇( P＜0.05)。EWS 306組腫瘤的體積(978.9±28.2) mm3明顯小于陰性對照組(1992.9±16.1) mm3和空白對照組(2001.9±12.3) mm3( P＜0.05)。接種後第35天，EWS 306組存活率為80%大于空白對照組為0%、陰性對照組為10%( P＜0.05)；接種後第40天，空白對照組降為0%， EWS306組為80%明顯大于和2箇對照組(P＜0.05)。結論 EWS/FLI-1蛋白錶位肽中EWS306較EWS289和EWS 401對DC的抗腫瘤作用更彊，能夠有效激活CTLs對腫瘤細胞的殺傷效應，為尤文肉瘤的免疫治療提供瞭新的方嚮。
목적비교불동EWS/FLI-1단백HLA-A/0201한제성표위태대우문육류수돌상세포(dendriticcell，DC)역묘적항종류작용。방법종합운용BIMAS、SYFPEITHI연건사선여인백세포항원HLA-A/0201결합력강적EWS/FLI-1단백9태표위，병합성기표위태。채용4 h표준51Cr석방실험검측자격세포독성T림파세포( cytotoxic T lymphocytes，CTLs )대종류세포적살상효응。응용매련면역반점법( enzyme linked immunospot assay，ELISPOT )검측표위태대DC역묘자격적효응세포γ-간우소( interferon，IFN-γ)적석방。재차기출상，진행면역치료적동물실험，비교기체내항종류작용。결과사선출EWS306、EWS289급EWS 401여HLA-A/0201구교강적결합성，기합성출적표위태분별위：QLWQFLLEL ( EWS 306)、ILGPTSSRL ( EWS 289)화SMYKYPSDI ( EWS 401)。3조표위태균유교강적살상력，당효-파비위50∶1시，살상솔EWS 306조(20.2±1.8)%、EWS 289조(12.6±0.3)%、EWS 401조(11.9±0.2)%，이대조조위(6.7±0.1)%；당효-파비위100∶1시，표위태적삼조살상작용명현제승，살상솔EWS 306조최고위(51.2±3.7)%、EWS 289조(24.6±2.1)%、EWS 401조(17.8±0.9)%，대조조위(7.2±0.2)%。EWS 306조동기타조비교，차이유통계학( P＜0.05)。EWS 306조분비적IFN-γ위(118.3±3.6)개점명현고우EWS401조(35.1±1.0)개화EWS 289조(34.2±0.9)개，대조조위(5.0±0.1)개( P＜0.05)。EWS 306조종류적체적(978.9±28.2) mm3명현소우음성대조조(1992.9±16.1) mm3화공백대조조(2001.9±12.3) mm3( P＜0.05)。접충후제35천，EWS 306조존활솔위80%대우공백대조조위0%、음성대조조위10%( P＜0.05)；접충후제40천，공백대조조강위0%， EWS306조위80%명현대우화2개대조조(P＜0.05)。결론 EWS/FLI-1단백표위태중EWS306교EWS289화EWS 401대DC적항종류작용경강，능구유효격활CTLs대종류세포적살상효응，위우문육류적면역치료제공료신적방향。
Objective To compare the antitumor effects of Ewing’s sarcoma protein ( EWS ) 306, EWS 289 and EWS 401 of human leukocyte antigen ( HLA )-antigen ( A ) / 0201-restricted epitope peptides against dendritic cells ( DCs ) of Ewing’s sarcoma.Methods The peptide 9 of EWS / FLI-1 protein, which was easily combined with HLA-A / 0201 of human cell antigen was screened by Operating software of BIMAS and SYFPEITHI. And then the corresponding epitope peptide was generated. The standard 4 h-51Cr release experiment was used to test the lethal effects of cytotoxic T lymphocytes ( CTLs ) against tumor cells. Enzyme linked immunospot assay ( ELISPOT ) was applied to detect the interferon-γ ( IFN-γ ) releasing of effector cells induced by DC vaccines of epitope peptides. Accordingly, the antitumor effects in vitro were compared in an animal experiment, and all these animals received immunotherapy. Results A strong binding force with HLA-A / 0201 was noticed in the screened EWS 306, EWS 289 and EWS 401, and the generated epitope peptides were QLWQFLLEL ( EWS 306 ), ILGPTSSRL ( EWS 289 ) and SMYKYPSDI ( EWS 401 ) respectively. A high lethality was noticed in EWS 306, EWS 289 and EWS 401 groups. When the effector /target cell ratio was 50 : 1, the lethalities in EWS 306, EWS 289 and EWS 401 groups were ( 20.2±1.8 ) %, ( 12.6±0.3 ) % and ( 11.9±0.2 ) % respectively. And while in the control group, the lethality was ( 6.7±0.1 ) %. When the effector / target cell ratio was 100:1, the lethalities in EWS 306, EWS 289, EWS 401 and the control groups were ( 51.2±3.7 ) %, ( 24.6±2.1 ) %, ( 17.8±0.9 ) % and ( 7.2±0.2 ) % respectively. There were statistically signiifcant differences when the lethality between the EWS 306 group and the others was compared (P<0.05 ). The number of secreted IFN-γ in EWS 306 group was 118.3±3.6, which was obviously higher than 35.1±1.0 in EWS 401 group, 34.2±0.9 in EWS 289 group and 5.0±0.1 in the control group ( P<0.05 ). The tumor volume was ( 978.9±28.2 ) mm3 in EWS 306 group, which was obviously smaller than ( 1992.9±16.1 ) mm3 in the negative control group and ( 2001.9±12.3 ) mm3 in the blank control group. At the 35 day after inoculation, the survival rate in EWS 306 group was 80%, which was higher than 0% in the blank control group and 10% in the negative control group (P<0.05 ). At 40 day after inoculation, the survival rate in the blank control group was reduced to 0%, and 80% in EWS 306 group was obviously higher than that of the other control groups (P<0.05 ).Conclusions Stronger antitumor effects against DCs are noticed in EWS 306 when compared with that of EWS 289 and EWS 401. EWS 306 can effectively activate the lethal effects of CTLs against tumor cells and provide a new direction for the immunotherapy for Ewing's sarcoma.
