目的 探讨人乳头状瘤病毒(HPV)16型多肽疫苗的制备,并观察HPV16多肽疫苗的体内外效应.方法 (1)针对抗原加工相关转运子(TAP)设计HPV16 E7蛋白的主要组织相容性复合物Ⅰ类分子(MHC-I)的抗原结合表位,利用生物信息学分析平台筛选出一致性较高、特异性及亲和力较强的HPV16 E7多肽作为研究对象制备HPV16多肽疫苗用于以下研究,本研究共筛选出3段多肽,分别命名为E7Pa、E7Pb、E7Pc.(2)C57BL/6小鼠注射鼠肺上皮细胞株TC-1细胞(为鼠源性的HPV16阳性的肿瘤细胞株)后,采用等额抽取的随机方法分为5组,ETPa+二核苷胞嘧啶(CpG)、E7Pb+CpG、E7Pc+CpG[均为实验组,分别加入终浓度为50μg/ml的E7Pa、E7Pb、E7Pc和终浓度为12 mg/L的刀豆蛋白(ConA)]、CpG(为阳性对照,加入终浓度为12 mg/L 的Con A)和空白对照组(不做任何处理).采用四甲基偶氮唑蓝(MTT)比色法检测各组作用不同时间后小鼠脾T淋巴细胞的体外增殖效应,乳酸脱氢酶(LDH)释放法检测小鼠脾T淋巴细胞在不同效靶比下的体外细胞毒T淋巴细胞(CTL)活性,实时荧光定量RT-PER技术检测小鼠肿瘤组织中γ干扰素(IFN-γ)、白细胞介素2(IL-2)mRNA的表达水平,酶联免疫吸附试验(ELISA)检测小鼠外周血中IFN-γ、IL-2的表达水平,通过定期测量比较各组小鼠接种HPV16多肽疫苗后体内肿瘤体积的变化.结果 (1)本研究筛选出了一致性较高、特异性及亲和力较强的3段HPV16 E7多肽作为研究对象制备HPV16多肽疫苗,分别命名为E7Pa、E7Pb、E7Pc.(2)MTT比色法检测显示,在接种疫苗24、48、72、96 h后,以E7Pa+CpG组的增殖效应最明显,其细胞增殖率分别为(131±32)%、(302±15)%、(552±28)%、(731±24)%,明显高于空白对照组的(72±15)%、(120±57)%、(176±41)%、(288±29)%(P<0.01);E7Pb+CpG、ETPc+CpG、CpG组的细胞增殖率也均明显高于空白对照组(P<0.05);但E7Pb+CpG、E7Pc+CpG、CpG组间比较,差异则无统计学意义(P>0.05).LDH释放法榆测显示,效靶比为100:1时,E7Pa+CpG、E7Pb+CpG、E7Pc+CpG、CpG和空白对照组CTL 活性分别为(85.9±3.0)%、(55.9±2.5)%、(60.2±1.5)%、(41.0±1.7)%和(4.1±1.0)%,E7Pa+CpG组与空白对照组比较,差异有统计学意义(P<0.01);E7Pb+CpG、E7Pc+CpG、CpG组分别与空白对照组比较,差异也有统计学意义(P<0.05);但E7Pb+CpG、E7Pc+CpG、CpG组间比较,差异则无统计学意义(P>0.05).在肿瘤组织及外周血中,小鼠IFN-γ、IL-2的表达水平,E7Pa+CpG组与空白对照组比较,差异均有统计学意义(P<0.01);E7Pb+CpG、E7Pc+CpG和CpG组分别与空白对照组比较,差异也均有统计学意义(P<0.05);但E7Pb+CpG、E7Pc+CpG、CpG组问比较,差异则无统计学意义(P>0.05).小鼠体内的肿瘤体积,各实验组肿瘤生长均明显被抑制,接种后第60大,E7Pa+CpG组与空白对照组比较,差异有统计学意义(P<0.01);E7Pb+CpG、E7Pc+CpG和CpG组分别与空白对照组比较,差异也均有统计学意义(P<0.05);但 E7Pb+CpG、E7Pc+CpG、CpG组间比较,差异则无统计学意义(P>0.05).结论 在动物模型中,针对TAP筛选的HPV16 E7多肽联合CpG制备的HPV16多肽疫苗,可以有效治疗HPV16 E7阳性的肿瘤.
目的 探討人乳頭狀瘤病毒(HPV)16型多肽疫苗的製備,併觀察HPV16多肽疫苗的體內外效應.方法 (1)針對抗原加工相關轉運子(TAP)設計HPV16 E7蛋白的主要組織相容性複閤物Ⅰ類分子(MHC-I)的抗原結閤錶位,利用生物信息學分析平檯篩選齣一緻性較高、特異性及親和力較彊的HPV16 E7多肽作為研究對象製備HPV16多肽疫苗用于以下研究,本研究共篩選齣3段多肽,分彆命名為E7Pa、E7Pb、E7Pc.(2)C57BL/6小鼠註射鼠肺上皮細胞株TC-1細胞(為鼠源性的HPV16暘性的腫瘤細胞株)後,採用等額抽取的隨機方法分為5組,ETPa+二覈苷胞嘧啶(CpG)、E7Pb+CpG、E7Pc+CpG[均為實驗組,分彆加入終濃度為50μg/ml的E7Pa、E7Pb、E7Pc和終濃度為12 mg/L的刀豆蛋白(ConA)]、CpG(為暘性對照,加入終濃度為12 mg/L 的Con A)和空白對照組(不做任何處理).採用四甲基偶氮唑藍(MTT)比色法檢測各組作用不同時間後小鼠脾T淋巴細胞的體外增殖效應,乳痠脫氫酶(LDH)釋放法檢測小鼠脾T淋巴細胞在不同效靶比下的體外細胞毒T淋巴細胞(CTL)活性,實時熒光定量RT-PER技術檢測小鼠腫瘤組織中γ榦擾素(IFN-γ)、白細胞介素2(IL-2)mRNA的錶達水平,酶聯免疫吸附試驗(ELISA)檢測小鼠外週血中IFN-γ、IL-2的錶達水平,通過定期測量比較各組小鼠接種HPV16多肽疫苗後體內腫瘤體積的變化.結果 (1)本研究篩選齣瞭一緻性較高、特異性及親和力較彊的3段HPV16 E7多肽作為研究對象製備HPV16多肽疫苗,分彆命名為E7Pa、E7Pb、E7Pc.(2)MTT比色法檢測顯示,在接種疫苗24、48、72、96 h後,以E7Pa+CpG組的增殖效應最明顯,其細胞增殖率分彆為(131±32)%、(302±15)%、(552±28)%、(731±24)%,明顯高于空白對照組的(72±15)%、(120±57)%、(176±41)%、(288±29)%(P<0.01);E7Pb+CpG、ETPc+CpG、CpG組的細胞增殖率也均明顯高于空白對照組(P<0.05);但E7Pb+CpG、E7Pc+CpG、CpG組間比較,差異則無統計學意義(P>0.05).LDH釋放法榆測顯示,效靶比為100:1時,E7Pa+CpG、E7Pb+CpG、E7Pc+CpG、CpG和空白對照組CTL 活性分彆為(85.9±3.0)%、(55.9±2.5)%、(60.2±1.5)%、(41.0±1.7)%和(4.1±1.0)%,E7Pa+CpG組與空白對照組比較,差異有統計學意義(P<0.01);E7Pb+CpG、E7Pc+CpG、CpG組分彆與空白對照組比較,差異也有統計學意義(P<0.05);但E7Pb+CpG、E7Pc+CpG、CpG組間比較,差異則無統計學意義(P>0.05).在腫瘤組織及外週血中,小鼠IFN-γ、IL-2的錶達水平,E7Pa+CpG組與空白對照組比較,差異均有統計學意義(P<0.01);E7Pb+CpG、E7Pc+CpG和CpG組分彆與空白對照組比較,差異也均有統計學意義(P<0.05);但E7Pb+CpG、E7Pc+CpG、CpG組問比較,差異則無統計學意義(P>0.05).小鼠體內的腫瘤體積,各實驗組腫瘤生長均明顯被抑製,接種後第60大,E7Pa+CpG組與空白對照組比較,差異有統計學意義(P<0.01);E7Pb+CpG、E7Pc+CpG和CpG組分彆與空白對照組比較,差異也均有統計學意義(P<0.05);但 E7Pb+CpG、E7Pc+CpG、CpG組間比較,差異則無統計學意義(P>0.05).結論 在動物模型中,針對TAP篩選的HPV16 E7多肽聯閤CpG製備的HPV16多肽疫苗,可以有效治療HPV16 E7暘性的腫瘤.
목적 탐토인유두상류병독(HPV)16형다태역묘적제비,병관찰HPV16다태역묘적체내외효응.방법 (1)침대항원가공상관전운자(TAP)설계HPV16 E7단백적주요조직상용성복합물Ⅰ류분자(MHC-I)적항원결합표위,이용생물신식학분석평태사선출일치성교고、특이성급친화력교강적HPV16 E7다태작위연구대상제비HPV16다태역묘용우이하연구,본연구공사선출3단다태,분별명명위E7Pa、E7Pb、E7Pc.(2)C57BL/6소서주사서폐상피세포주TC-1세포(위서원성적HPV16양성적종류세포주)후,채용등액추취적수궤방법분위5조,ETPa+이핵감포밀정(CpG)、E7Pb+CpG、E7Pc+CpG[균위실험조,분별가입종농도위50μg/ml적E7Pa、E7Pb、E7Pc화종농도위12 mg/L적도두단백(ConA)]、CpG(위양성대조,가입종농도위12 mg/L 적Con A)화공백대조조(불주임하처리).채용사갑기우담서람(MTT)비색법검측각조작용불동시간후소서비T림파세포적체외증식효응,유산탈경매(LDH)석방법검측소서비T림파세포재불동효파비하적체외세포독T림파세포(CTL)활성,실시형광정량RT-PER기술검측소서종류조직중γ간우소(IFN-γ)、백세포개소2(IL-2)mRNA적표체수평,매련면역흡부시험(ELISA)검측소서외주혈중IFN-γ、IL-2적표체수평,통과정기측량비교각조소서접충HPV16다태역묘후체내종류체적적변화.결과 (1)본연구사선출료일치성교고、특이성급친화력교강적3단HPV16 E7다태작위연구대상제비HPV16다태역묘,분별명명위E7Pa、E7Pb、E7Pc.(2)MTT비색법검측현시,재접충역묘24、48、72、96 h후,이E7Pa+CpG조적증식효응최명현,기세포증식솔분별위(131±32)%、(302±15)%、(552±28)%、(731±24)%,명현고우공백대조조적(72±15)%、(120±57)%、(176±41)%、(288±29)%(P<0.01);E7Pb+CpG、ETPc+CpG、CpG조적세포증식솔야균명현고우공백대조조(P<0.05);단E7Pb+CpG、E7Pc+CpG、CpG조간비교,차이칙무통계학의의(P>0.05).LDH석방법유측현시,효파비위100:1시,E7Pa+CpG、E7Pb+CpG、E7Pc+CpG、CpG화공백대조조CTL 활성분별위(85.9±3.0)%、(55.9±2.5)%、(60.2±1.5)%、(41.0±1.7)%화(4.1±1.0)%,E7Pa+CpG조여공백대조조비교,차이유통계학의의(P<0.01);E7Pb+CpG、E7Pc+CpG、CpG조분별여공백대조조비교,차이야유통계학의의(P<0.05);단E7Pb+CpG、E7Pc+CpG、CpG조간비교,차이칙무통계학의의(P>0.05).재종류조직급외주혈중,소서IFN-γ、IL-2적표체수평,E7Pa+CpG조여공백대조조비교,차이균유통계학의의(P<0.01);E7Pb+CpG、E7Pc+CpG화CpG조분별여공백대조조비교,차이야균유통계학의의(P<0.05);단E7Pb+CpG、E7Pc+CpG、CpG조문비교,차이칙무통계학의의(P>0.05).소서체내적종류체적,각실험조종류생장균명현피억제,접충후제60대,E7Pa+CpG조여공백대조조비교,차이유통계학의의(P<0.01);E7Pb+CpG、E7Pc+CpG화CpG조분별여공백대조조비교,차이야균유통계학의의(P<0.05);단 E7Pb+CpG、E7Pc+CpG、CpG조간비교,차이칙무통계학의의(P>0.05).결론 재동물모형중,침대TAP사선적HPV16 E7다태연합CpG제비적HPV16다태역묘,가이유효치료HPV16 E7양성적종류.
Objective To prepare the human papillomavirus (HPV) 16 peptide vaccine and explore the effect in vitro and in vivo. Methods (1) Prediction of the major histocompatibility complex (MHC) class I restricted T cell epitopes by bioinformatics target at transporter associated with antigen processing (TAP) and named by E7Pa, E7Pb, E7Pc separately. (2)In vivo, the C57BL/6 mice were divided into five groups with same amounts randomly after loading with TC-1 cells (HPV 16 positive tumor cells from C57BL/6 mouse), named as E7Pa + CpG,E7Pb + CpG,E7Pc + CpG (as experiment groups, and added 50 μg/ml E7Pa, E7Pb, E7Pc, respectively), CpG(as positive control group and added Con A with 12 mg/L final concentration) and blank control group (without any treatment). The T cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) assay at different time points;the lactate dehydrogenase (LDH) delivery method was used to test the cytolytie T lymphocyte (CTL) activity of mouse splenic lymphocyte in different ratio of effector cells and target cells (E:T);the related cytokines in tumor tissue and mouse peripheral blood were evaluated by real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. The tumor volumes were measured to contrast the therapeutic effect in different groups. Results (1) Three peptide named E7Pa, E7Pb, E7Pc were successfully preparated which had high affinity and specificity. (2) After vaccination of 24, 48, 72,96 hours, MTT results shown that the proliferation rate in E7Pa + CpG group were(131±32)%, (302±15)%, (552±28)%, (731±24)% individually, which were much higher than those in blank control [(72± 15) %, (120 ± 57) %, (176 ±41)%, (288±29)% ;P<0.01], and the other groups i. e. E7Pb + CpG,E7Pc +CpG and CpG groups all proliferated much higher than those in blank control group with statistic signification (P<0. 05), but there was no significant difference between groups(P>0.05);the LDH delivery assay showed that when the ratio of E:T was 100:1, the activity of CTL in the E7Pa + CpG group was most powerful than the other groups with statistic signification (P<0. 01). Meanwhile, the ratio of E:T was concentration-dependent. Compared E7Pb + CpG, E7Pc + CpG or CpG groups with blank control group, there were significantly difference(P<0. 05) ,while there was no significant difference between groups(P >0. 05). The mRNA levels of interferon γ (IFN-γ), interleukin-2 (IL-2) in tumor tissue and peripheral blood in E7Pa + CpG group were significantly higher than those in blank control group (P<0. 01), which was the similar results when compared E7Pb + CpG, E7Pc + CpG or CpG groups with control group (P < 0. 05), and without significant difference between groups(P > 0. 05). The tumor volumes were suppressed obviously in all the experiment groups, especially at the 60th days, the volumes in ETPa + CpG group were much smaller than that in blank control group with statistic signification (P < 0. 01),which was the similar results that E7Pb + CpG, E7Pc + CpG or CpG groups had difference than blank control group with statistic signification (P < 0. 05), and without significant difference between groups(P >0. 05). Conclusion The HPV16 E7 peptide target at TAP combination with CpG as a vaccine could treat effectively the HPV16 E7 positive tumor in experiment.
