中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2012年
5期
468-472
,共5页
浦红%史央%陆英华%朱晨瑶%毛玉荣%张振宇%时宏珍
浦紅%史央%陸英華%硃晨瑤%毛玉榮%張振宇%時宏珍
포홍%사앙%륙영화%주신요%모옥영%장진우%시굉진
人乳头瘤病毒%多肽%树突状细胞%细胞毒性T细胞
人乳頭瘤病毒%多肽%樹突狀細胞%細胞毒性T細胞
인유두류병독%다태%수돌상세포%세포독성T세포
Human papillomavirus%Peptide%Dendritic cell%Cytotoxic T lymphocytes
目的 观察以高危型人乳头瘤病毒16型(HPV16) E6和E7基因编码的多肽E613-21(KLPDLCTEL)和E786-94(TLGIVCPI)为抗原负载的树突状细胞(DC)体外诱导特异性CTL的能力.方法 采集HPV+HLA-A2+宫颈癌患者外周血,分离单核细胞(Mo)与外周淋巴细胞(PBL)；将Mo诱导成DC后负载E6和E7多肽,用其反复致敏PBL(3次)；ELISA法检测致敏细胞上清细胞因子分泌水平；流式细胞标记法检测致敏细胞中特异性CTL的比例；MTT法检测致敏细胞杀伤靶细胞的能力.结果 11例HPV 16+ HLA-A2+宫颈癌患者DC平均得率为(10.79±0.88) ×l06(每100 ml外周血),CD11c+ HLA-DR+为(97.15±2.41)％,其中CD80+为(84.28±5.39)％,CD83+为(85.17+5.06)％,CD86+为(97.74±0.87)％.PBLs致敏3次后,平均增殖(15.4±1.5)倍；致敏第21天上清细胞因子水平分别为:IL-2(2551.9±195.3) pg/ml、IL-12(554.9士64.0)pg/ml、IFN-γ(2416.9±281.7)pg/ml,TNF-α(632.4±71.1) pg/ml,明显高于未致敏组(P＜0.05),IL-10(235.1+34.7) pg/ml与对照组比较无显著差异；特异性CTL的平均比例为(6.32±1.54)％,明显高于对照组(P＜0.05),对负载E6、E7多肽的T2细胞株杀伤率明显高于对照组(P＜0.05).结论 HPV E6和E7混合多肽负载的DC体外能有效地诱导特异性CTL,刺激Thl型细胞因子的分泌,为治疗型宫颈癌疫苗的研制提供科学依据.
目的 觀察以高危型人乳頭瘤病毒16型(HPV16) E6和E7基因編碼的多肽E613-21(KLPDLCTEL)和E786-94(TLGIVCPI)為抗原負載的樹突狀細胞(DC)體外誘導特異性CTL的能力.方法 採集HPV+HLA-A2+宮頸癌患者外週血,分離單覈細胞(Mo)與外週淋巴細胞(PBL)；將Mo誘導成DC後負載E6和E7多肽,用其反複緻敏PBL(3次)；ELISA法檢測緻敏細胞上清細胞因子分泌水平；流式細胞標記法檢測緻敏細胞中特異性CTL的比例；MTT法檢測緻敏細胞殺傷靶細胞的能力.結果 11例HPV 16+ HLA-A2+宮頸癌患者DC平均得率為(10.79±0.88) ×l06(每100 ml外週血),CD11c+ HLA-DR+為(97.15±2.41)％,其中CD80+為(84.28±5.39)％,CD83+為(85.17+5.06)％,CD86+為(97.74±0.87)％.PBLs緻敏3次後,平均增殖(15.4±1.5)倍；緻敏第21天上清細胞因子水平分彆為:IL-2(2551.9±195.3) pg/ml、IL-12(554.9士64.0)pg/ml、IFN-γ(2416.9±281.7)pg/ml,TNF-α(632.4±71.1) pg/ml,明顯高于未緻敏組(P＜0.05),IL-10(235.1+34.7) pg/ml與對照組比較無顯著差異；特異性CTL的平均比例為(6.32±1.54)％,明顯高于對照組(P＜0.05),對負載E6、E7多肽的T2細胞株殺傷率明顯高于對照組(P＜0.05).結論 HPV E6和E7混閤多肽負載的DC體外能有效地誘導特異性CTL,刺激Thl型細胞因子的分泌,為治療型宮頸癌疫苗的研製提供科學依據.
목적 관찰이고위형인유두류병독16형(HPV16) E6화E7기인편마적다태E613-21(KLPDLCTEL)화E786-94(TLGIVCPI)위항원부재적수돌상세포(DC)체외유도특이성CTL적능력.방법 채집HPV+HLA-A2+궁경암환자외주혈,분리단핵세포(Mo)여외주림파세포(PBL)；장Mo유도성DC후부재E6화E7다태,용기반복치민PBL(3차)；ELISA법검측치민세포상청세포인자분비수평；류식세포표기법검측치민세포중특이성CTL적비례；MTT법검측치민세포살상파세포적능력.결과 11례HPV 16+ HLA-A2+궁경암환자DC평균득솔위(10.79±0.88) ×l06(매100 ml외주혈),CD11c+ HLA-DR+위(97.15±2.41)％,기중CD80+위(84.28±5.39)％,CD83+위(85.17+5.06)％,CD86+위(97.74±0.87)％.PBLs치민3차후,평균증식(15.4±1.5)배；치민제21천상청세포인자수평분별위:IL-2(2551.9±195.3) pg/ml、IL-12(554.9사64.0)pg/ml、IFN-γ(2416.9±281.7)pg/ml,TNF-α(632.4±71.1) pg/ml,명현고우미치민조(P＜0.05),IL-10(235.1+34.7) pg/ml여대조조비교무현저차이；특이성CTL적평균비례위(6.32±1.54)％,명현고우대조조(P＜0.05),대부재E6、E7다태적T2세포주살상솔명현고우대조조(P＜0.05).결론 HPV E6화E7혼합다태부재적DC체외능유효지유도특이성CTL,자격Thl형세포인자적분비,위치료형궁경암역묘적연제제공과학의거.
Objective To explore the potential of autologous dendritic cells (DC) pulsed with HLA-A201-binding peptide E613-21(KLPDLCTEL) and E786-94(TLGIVCPI)in inducing specific T cells respouse in vitro.Methods Cervical carcinoma patients with positive HLA-A201 were enrolled and their monocytes isolated and induced into dendritic cells and pulsed with HLA-A201-binding peptide E613-21 and E786-94.PBLs were primed by DCs every week for thee times.The cytokine level of supernatant of CTLs was tested by ELISA.The percentage of special CTLs was tested by flow cytometry.The specific killing effect of CTLs was tested by MTT.Results the numbers of DCs of eleven cervical carcinoma patients were (10.79±0.88) ×106(100 ml peripheral blood).CDllc+HLA-DR+(97.15±2.41)％,CD80+(84.28+5.39)％,CD83 +(85.17±5.06) ％,CD86 + (97.74+0.87) ％.Proliferation index of PBLs primed by DCs three times was 15.4± 1.5.Cytokine levels including IL-2,IL-12,IFN-γ and TNF-α were obviously higher than nonpriming PBLs[(2551.9+195.3) pg/ml,(554.9±64.0) pg/ml,(2416.9±281.7) pg/ml,(632.4 +71.1)pg/ml,respectively] (P＜0.05),but IL-10 was no significant difference between priming CTLs and nonpriming CTLs.The average percentage of special CTLs was obviously higher than control group[(6.32±1.54)％,P＜0.05].The killing effect of CTLs was obviously higher than control group(P＜0.05).Conclusion Dendritic cells pulsed with peptide E613-21 and E786-94 can induce special CTLs in vitro and stimulate CTLs secret cytokines.This will provide science basis for research of therapeutic HPV vaccine.
