本文選題：五峰縣 + 人乳頭瘤病毒��； 參考：《南昌大學(xué)》2014年碩士論文

【摘要】：目的： 初步研究湖北五峰土家族婦女人乳頭瘤病毒16型E7抗原限制性細(xì)胞毒性T淋巴細(xì)胞表位，為該地區(qū)遠(yuǎn)期表位抗原的特異性鑒定和臨床研究提供靶肽。 方法： 使用NetMHCpan軟件預(yù)測HPV16型E7抗原CTL表位，采用標(biāo)準(zhǔn)的Fmoc方案合成多肽，產(chǎn)物采用RP-HPLC純化、分析，并進(jìn)行質(zhì)譜分析鑒定；選取湖北五峰縣土家族健康婦女和原發(fā)性宮頸癌各30名并提取外周血DNA，使用按照優(yōu)諾基HLA-SSO流式熒光微珠基因分型試劑盒進(jìn)行HLA分型，篩選出HLA-A*02+健康婦女和HLA-B*07+宮頸癌患者各5名，采集外周血單個核細(xì)胞（PBMCs）用E7肽體外刺激進(jìn)行免疫學(xué)實(shí)驗(yàn)，采用ELISOT技術(shù)檢測IFN-γ斑點(diǎn)形成細(xì)胞數(shù)（Spots Forming Cells，SFCs）水平的差異并進(jìn)行統(tǒng)計(jì)學(xué)分析。 結(jié)果 經(jīng)RP-HPLC純化后，多肽產(chǎn)物純度均在95%以上，采用質(zhì)譜法鑒定出多肽實(shí)測分子量與理論值相吻合，鑒定為高純度的目標(biāo)肽。 E7肽體外刺激HLA-A*02+健康人PBMCs誘導(dǎo)機(jī)體產(chǎn)生了顯著的CTLs反應(yīng)，IFN-γ斑點(diǎn)形成細(xì)胞數(shù)目明顯。組間差異用方差分析，組間差異有統(tǒng)計(jì)學(xué)意義（F=15.17，P＜0.001），，兩兩比較進(jìn)行LSD檢驗(yàn)，與陰性對照組比較，實(shí)驗(yàn)組差異均有統(tǒng)計(jì)學(xué)意義，其中以P2:E741-55、P4:E751-65差異最顯著（P＜0.05），二者無顯著性差異。 E7肽體外刺激HLA-B*07+宮頸癌患者PBMCs未能誘導(dǎo)機(jī)體產(chǎn)生顯著的CTLs反應(yīng)，INF-γ斑點(diǎn)形成細(xì)胞數(shù)目少，組間差異用方差分析，組間差異有統(tǒng)計(jì)學(xué)意義（F=40.26，P＜0.001），兩兩比較進(jìn)行LSD檢驗(yàn)，與陰性對照組比較，以P5：E766-80差異最顯著，且有統(tǒng)計(jì)學(xué)意義（P＜0.05）。 HLA-A*02+健康人與HLA-B*07+宮頸癌患者ELISPOT結(jié)果提示INF-r斑點(diǎn)形成細(xì)胞數(shù)目有明顯差異，且差異性有統(tǒng)計(jì)學(xué)意義（F=268.82，P＜0.001）。 對不同多肽誘導(dǎo)機(jī)體產(chǎn)生CTLs反應(yīng)結(jié)果進(jìn)行SNK檢驗(yàn)，P2:E741-55、P4:E751-65和P5:E766-80三條多肽對結(jié)果的影響最明顯，與其他多肽比較差異有統(tǒng)計(jì)學(xué)意義（P0.05），但三者間尚不能認(rèn)為有統(tǒng)計(jì)學(xué)差異。 結(jié)論： HLA-A*02能有效呈遞HPV16型E7抗原肽誘導(dǎo)機(jī)體產(chǎn)生顯著性CTLs反應(yīng)；HLA-B*07不能有效呈遞HPV16型E7抗原肽誘導(dǎo)機(jī)體產(chǎn)生顯著性CTLs反應(yīng)；E741-55及E751-65具有較強(qiáng)抗原性，初步篩選為HLA-A*02限制性CTL候選表位肽。
[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- 緯 speckle forming cells (IFN- 緯) 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- 緯 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- 緯 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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.33

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