本文選題：五峰縣 + 人乳頭瘤病毒　； 參考：《南昌大學》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抗原限制性細胞毒性T淋巴細胞表位，為該地區(qū)遠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軟件預測HPV16型E7抗原CTL表位，采用標準的Fmoc方案合成多肽，產(chǎn)物采用RP-HPLC純化、分析，并進行質(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胞（PBMCs）用E7肽體外刺激進行免疫學實驗，采用ELISOT技術檢測IFN-γ斑點形成細胞數(shù)（Spots Forming Cells，SFCs）水平的差異并進行統(tǒng)計學分析。 結果 經(jīng)RP-HPLC純化后，多肽產(chǎn)物純度均在95%以上，采用質(zhì)譜法鑒定出多肽實測分子量與理論值相吻合，鑒定為高純度的目標肽。 E7肽體外刺激HLA-A*02+健康人PBMCs誘導機體產(chǎn)生了顯著的CTLs反應，IFN-γ斑點形成細胞數(shù)目明顯。組間差異用方差分析，組間差異有統(tǒng)計學意義（F=15.17，P＜0.001），，兩兩比較進行LSD檢驗，與陰性對照組比較，實驗組差異均有統(tǒng)計學意義，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未能誘導機體產(chǎn)生顯著的CTLs反應，INF-γ斑點形成細胞數(shù)目少，組間差異用方差分析，組間差異有統(tǒng)計學意義（F=40.26，P＜0.001），兩兩比較進行LSD檢驗，與陰性對照組比較，以P5：E766-80差異最顯著，且有統(tǒng)計學意義（P＜0.05）。 HLA-A*02+健康人與HLA-B*07+宮頸癌患者ELISPOT結果提示INF-r斑點形成細胞數(shù)目有明顯差異，且差異性有統(tǒng)計學意義（F=268.82，P＜0.001）。 對不同多肽誘導機體產(chǎn)生CTLs反應結果進行SNK檢驗，P2:E741-55、P4:E751-65和P5:E766-80三條多肽對結果的影響最明顯，與其他多肽比較差異有統(tǒng)計學意義（P0.05），但三者間尚不能認為有統(tǒng)計學差異。 結論： HLA-A*02能有效呈遞HPV16型E7抗原肽誘導機體產(chǎn)生顯著性CTLs反應；HLA-B*07不能有效呈遞HPV16型E7抗原肽誘導機體產(chǎn)生顯著性CTLs反應；E741-55及E751-65具有較強抗原性，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.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.33

【參考文獻】

相關期刊論文 前10條

1 吳玉章;免疫識別、分子設計與抗原工程[J];第三軍醫(yī)大學學報;2000年10期

2 宋云煥;李莉;;四價HPV疫苗預防宮頸癌、HPV感染相關疾病Meta分析[J];國際婦產(chǎn)科學雜志;2009年06期

3 朱錫華,吳玉章;對表位生物學研究的認識和體會[J];上海免疫學雜志;1998年01期

4 宋云煥;周自廣;;二價HPV疫苗預防宮頸癌及HPV相關感染的meta分析[J];山西醫(yī)科大學學報;2012年05期

5 ;HLA-B*6701 as a Subtype Performing Post-Selected Marking Gene for Cervical Cancer[J];Wuhan University Journal of Natural Sciences;2010年06期

6 蔣衛(wèi);李寧麗;;人乳頭瘤病毒的免疫逃逸機制[J];細胞生物學雜志;2008年01期

7 王臨虹;邱t;鄭睿敏;狄江麗;;我國宮頸癌流行病學狀況及防治策略的回顧與展望[J];中國婦幼衛(wèi)生雜志;2010年03期

8 王又又;向群英;佘茜;譚云;劉忠;張蔚;陶寧;陳慶;魏蕓;伍欣星;;宮頸癌高發(fā)區(qū)婦女HPV感染及影響因素分析[J];中國公共衛(wèi)生;2011年03期

9 徐云升,郝飛,宋志強,鐘白玉,郝進,葉慶佾;HPV16E7抗原的HLA-A2限制性細胞毒性T淋巴細胞表位的篩選與鑒定[J];中華皮膚科雜志;2004年05期

10 邱小萍;譚云;左澤華;魏蕓;伍欣星;;湖北土家族人群HLA-A、B等位基因及單倍型多態(tài)性的分布[J];中華醫(yī)學遺傳學雜志;2006年02期

相關博士學位論文 前1條

1 尹銳;人乳頭瘤病毒16E7 HLA-A_2限制性CTL表位的穿膜肽疫苗設計及體外免疫學研究[D];第三軍醫(yī)大學;2006年

本文編號：1885082