湖北五峰土家族妇女HPV16型E7抗原限制性CTL表位的初步研究

发布时间：2018-05-13 22:22

本文选题：五峰县 + 人乳头瘤病毒　；参考：《南昌大学》2014年硕士论文

【摘要】：目的：初步研究湖北五峰土家族妇女人乳头瘤病毒16型E7抗原限制性细胞毒性T淋巴细胞表位，为该地区远期表位抗原的特异性鉴定和临床研究提供靶肽。方法：使用NetMHCpan软件预测HPV16型E7抗原CTL表位，采用标准的Fmoc方案合成多肽，产物采用RP-HPLC纯化、分析，并进行质谱分析鉴定；选取湖北五峰县土家族健康妇女和原发性宫颈癌各30名并提取外周血DNA，使用按照优诺基HLA-SSO流式荧光微珠基因分型试剂盒进行HLA分型，筛选出HLA-A*02+健康妇女和HLA-B*07+宫颈癌患者各5名，采集外周血单个核细胞（PBMCs）用E7肽体外刺激进行免疫学实验，采用ELISOT技术检测IFN-γ斑点形成细胞数（Spots Forming Cells，SFCs）水平的差异并进行统计学分析。结果经RP-HPLC纯化后，多肽产物纯度均在95%以上，采用质谱法鉴定出多肽实测分子量与理论值相吻合，鉴定为高纯度的目标肽。 E7肽体外刺激HLA-A*02+健康人PBMCs诱导机体产生了显著的CTLs反应，IFN-γ斑点形成细胞数目明显。组间差异用方差分析，组间差异有统计学意义（F=15.17，P＜0.001），，两两比较进行LSD检验，与阴性对照组比较，实验组差异均有统计学意义，其中以P2:E741-55、P4:E751-65差异最显著（P＜0.05），二者无显著性差异。 E7肽体外刺激HLA-B*07+宫颈癌患者PBMCs未能诱导机体产生显著的CTLs反应，INF-γ斑点形成细胞数目少，组间差异用方差分析，组间差异有统计学意义（F=40.26，P＜0.001），两两比较进行LSD检验，与阴性对照组比较，以P5：E766-80差异最显著，且有统计学意义（P＜0.05）。 HLA-A*02+健康人与HLA-B*07+宫颈癌患者ELISPOT结果提示INF-r斑点形成细胞数目有明显差异，且差异性有统计学意义（F=268.82，P＜0.001）。对不同多肽诱导机体产生CTLs反应结果进行SNK检验，P2:E741-55、P4:E751-65和P5:E766-80三条多肽对结果的影响最明显，与其他多肽比较差异有统计学意义（P0.05），但三者间尚不能认为有统计学差异。结论： HLA-A*02能有效呈递HPV16型E7抗原肽诱导机体产生显著性CTLs反应；HLA-B*07不能有效呈递HPV16型E7抗原肽诱导机体产生显著性CTLs反应；E741-55及E751-65具有较强抗原性，初步筛选为HLA-A*02限制性CTL候选表位肽。
[Abstract]:Objective: To study the restricted cytotoxic T lymphocyte epitopes of human papillomavirus type 16 (E7) antigen in Wufeng Tujia women in Hubei province, and to provide a target peptide for the identification and clinical study of the long term epitope antigen in this area. Methods: The CTL epitopes of HPV16 E7 antigen were predicted by NetMHCpan software. The peptides were synthesized by standard Fmoc scheme. The products were purified and analyzed by RP-HPLC and identified by mass spectrometry. The peripheral blood DNA was extracted from 30 healthy Tujia women and 30 primary cervical cancer women in Wufeng County, Hubei Province. The HLA typing kit was used according to Unoki HLA-SSO flow fluorescent microbead genotyping kit. Five healthy women with HLA-A*02 and 5 patients with HLA-B*07 cervical cancer were selected. Peripheral blood mononuclear cells (PBMCs) were collected and stimulated with E7 peptide in vitro for immunological experiment. The level of IFN- 纬 speckle forming cells (IFN- 纬) was detected by ELISOT technique and the difference of Spots Forming CellsCss was analyzed statistically. Result After purification by RP-HPLC, the purity of the polypeptide products was over 95%. The molecular weight of the polypeptide measured by mass spectrometry was in agreement with the theoretical value, and the peptide was identified as the target peptide with high purity. In vitro stimulation of PBMCs by E7 peptide in HLA-A*02 healthy subjects induced a significant CTLs response and the number of IFN- 纬 dot-forming cells was significant. The difference between the two groups was statistically significant (P < 0.001). Compared with the negative control group, the difference in the experimental group was statistically significant (P < 0.05), and the difference between the two groups was the most significant (P < 0.05), and there was no significant difference between the two groups (P < 0.05). The difference between the two groups was significant (P < 0.05), and there was no significant difference between the two groups in comparison with the negative control group (P < 0.05). In vitro stimulation of PBMCs with E7 peptide in HLA-B*07 patients with cervical cancer failed to induce a significant CTLs response. The number of INF- 纬 dot-forming cells was low. The difference among groups was statistically significant (P < 0.001), and the difference was statistically significant (P < 0.001). The difference between the two groups was tested by LSD. Compared with the negative control group, the difference of P5:E766-80 was the most significant (P < 0.05). The results of ELISPOT showed that there was significant difference in the number of INF-r dot-forming cells between healthy persons with HLA-A*02 and HLA-B*07 patients with cervical cancer, and the difference was statistically significant (P < 0.001). The results of CTLs reaction induced by different polypeptides were tested by SNK. The three polypeptides P21: E741-55, P4: E751-65 and P5:E766-80 had the most obvious effect on the results, and the differences were statistically significant compared with other polypeptides, but there was no statistical difference among the three polypeptides. Conclusion: HLA-A*02 can effectively present HPV16 type E7 antigen peptide to induce significant CTLs reaction. HLA-B7 antigen peptide can not effectively present HPV16 type E7 antigen peptide to induce body to produce significant CTLs reaction. E741-55 and E751-65 have strong antigenicity, so they are selected as HLA-A*02 restricted CTL candidate epitope peptides.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33

【参考文献】