人乳头瘤病毒分型及整合素抗体抑制尖锐湿疣复发的初步研究

发布时间：2018-07-12 20:39

本文选题：尖锐湿疣 + 复发　；参考：《天津医科大学》2011年硕士论文

【摘要】：尖锐湿疣(condyloma acuminatum, CA)是由人乳头瘤病毒(human papillomavirus, HPV)引起的,以发生在生殖器、肛周为主的性传播疾病(sexually transmitted disease, STD)。CA不仅发病率高,而且治疗后其复发率也很高。目前有多种针对CA的治疗方法,但任何一种方法都不能避免其复发。不同型的HPV感染可以引起不同的临床表现,对HPV进行分型将有利于临床疾病的诊断及预后的判定。整合素(integrin)是细胞黏附分子家族中重要的成员之一。通过大量对整合素与病毒感染关系的研究发现,多种病毒都能利用整合素作为病毒受体或共受体(co-receptor)进入宿主细胞。整合素α6主要在正常上皮组织基底层特异性表达,而HPV对宿主造成的损害,首先是以感染宿主上皮组织基底层为起始的,二者是否存在相关性?整合素α6在HPV感染过程中是否发挥作用?如果能在这个起始阶段将整合素的表达进行封闭,是否可使病毒对宿主细胞的黏附受到抑制?对这些问题的进一步研究将有助于从根本上阻断HPV病毒的感染和CA的复发。目的①鉴定和评估PCR联合限制性片段长度多态性(RFLP)及基因测序技术对CA及四种外生殖器和肛周皮肤病进行HPV DNA检测和分型的可行性；②检测CA患者皮损处整合素α6 mRNA的表达,初步探讨其在CA的发病与复发中的意义；③探讨整合素α6单克隆抗体(GoH3)对HPV 6/11病毒颗粒体外感染永生化人角质形成细胞株(HaCaT细胞)的影响。方法①应用PCR-RFLP-基因测序技术对发生于外生殖器和肛周部位的CA、鲍温样丘疹病(BP)、鳞状细胞癌(SCC)、硬化萎缩性苔藓(LSA)和乳房外Paget病(EMPD)的皮损组织进行HPV DNA检测和分型；②采用RT-PCR方法测定初发和复发CA患者皮损处整合素α6 mRNA的表达；③应用荧光定量聚合酶链反应(FQ-PCR)方法检测GoH3对临床CA组织中提取的HPV 6/病毒颗粒感染HaCaT细胞后HPV DNA载量的影响。结果①50例经病理确诊的CA及四种外生殖器和肛周皮肤病标本中,共有35例检测出HPV DNA,其阳性率分别为CA 100.0%(26/26)、BP 47.1%(8/17)、SCC 50.0%(1/2)、LSA 0.0%(0/4)和EMPD 0.0%(0/1); HPV DNA阳性标本中,HPV 6型19例,HPV-11型3例,HPV 16型8例,HPV 6型+11型4例,HPV 62型1例；基因测序结果与PCR-RFLP判定的HPV型别相符；②整合素α6在初发和复发CA皮损中.nRNA的相对病毒载量分别0.79±0.16和1.07±0.29,均高于正常包皮组织的0.25+0.10,差异有统计学意义(P=0.001)；复发CA皮损中整合素α6的表达高于初发,差异有统计学意义(P0.05)；③4组不同浓度的HPV 6/11病毒颗粒感染HaCaT细胞后测得的HPV病毒载量不同(P=0.001)。当病毒颗粒浓度为106copies/ml时,HaCaT细胞被感染率为100%,且所测得的HPV载量高于其它3组(P0.05)；当GoH3稀释倍数1：100时,其对HPV感染HaCaT细胞的抑制率与其浓度成正比；但当稀释倍数1：100时,再增加GoH3的浓度,其抑制率也未见明显增长；8例临床分离的HPV 6/11病毒颗粒在加入GoH3之前感染HaCaT细胞的平均病毒载量为(5.8±2.51)×104copies/ml,加入GoH3后感染HaCaT细胞的平均病毒载量为(3.02±1.21)×104copies/ml, GoH3的平均抑制率为(46.9±4.7)%。结论①PCR-RFLP方法可用于HPV DNA检测与分型；②整合素α6可能在CA发病和复发中发挥作用；③GoH3对HPV 6/11型感染HaCaT细胞具有一定的抑制作用。
[Abstract]:Condyloma acuminatum (CA) is caused by the human papillomavirus (human papillomavirus, HPV), which occurs in the genitals, and the sexually transmitted disease (sexually transmitted disease, STD) is not only high, but also has a high relapse rate after treatment. There are a variety of treatment methods for CA, but any one of them. The methods can not avoid the recurrence. Different types of HPV infection can cause different clinical manifestations. The classification of HPV will be beneficial to the diagnosis and prognosis of clinical diseases.
Integrin (integrin) is one of the most important members of the cell adhesion molecule family. Through a large number of studies on the relationship between integrin and virus infection, it is found that a variety of viruses can use integrin as a viral receptor or a common receptor (co-receptor) into the host cell. Integrin alpha 6 is mainly expressed in the basal layer of normal epithelial tissue, and HPV The damage to the host, first of all, begins with the infection of the basal layer of the epithelial tissue of the host. Is there a correlation between the two? Does the integrin alpha 6 play a role in the HPV infection? If the expression of the integrin is closed at this beginning, can the adhesion of the virus to the host cell be inhibited? Further studies will help to fundamentally block the infection of HPV virus and the recurrence of CA.
Objective (1) to identify and evaluate the feasibility of PCR combined restriction fragment length polymorphism (RFLP) and gene sequencing technology for HPV DNA detection and typing of CA and four kinds of external and perianal skin diseases. (2) the expression of integrin alpha 6 mRNA in the skin lesions of CA patients was detected and its significance in the pathogenesis and recurrence of CA was preliminarily discussed. The effect of monoclonal antibody GoH3 (HPV) on HPV 6/11 virus particles infecting immortalized human keratinocyte line (HaCaT cells) in vitro.
Methods (1) PCR-RFLP- gene sequencing was used to detect CA, bautherid papular disease (BP), squamous cell carcinoma (SCC), BP, LSA and Paget disease (EMPD) in the genital and perianal areas, and to detect and classify the lesions of the lesions of the skin tissues of Paget disease (EMPD). (2) the determination of integrin alpha at the skin lesions of primary and recurrence CA patients by RT-PCR method. 6 mRNA expression; (3) using fluorescence quantitative polymerase chain reaction (FQ-PCR) to detect the effect of GoH3 on the HPV DNA load of HPV 6/ virus infected HaCaT cells extracted from the clinical CA tissue.
Results (1) in 50 cases of CA and four types of external genitals and perianal skin diseases, there were 35 cases of HPV DNA detection, the positive rates were CA 100% (26/26), BP 47.1% (8/17), SCC 50% (1/2), LSA 0% (0/4) and EMPD 0%. The results of gene sequencing in 1 cases were consistent with the HPV type determined by PCR-RFLP, and the relative viral load of integrin alpha 6 in the primary and recurrent CA lesions was 0.79 + 0.16 and 1.07 + 0.29, respectively, higher than that of normal foreskin (P=0.001), and the expression of integrin alpha 6 in the recurrent CA lesions was higher than that of the initial hair. The difference was statistically significant (P0.05); (3) the HPV virus load of the 4 groups of different concentrations of HPV 6/11 virus particles infected with HaCaT cells was different (P=0.001). When the concentration of the virus particles was 106copies/ml, the infection rate of HaCaT cells was 100%, and the measured HPV load was higher than that of the other 3 groups (P0.05); when GoH3 dilution multiple 1:100, it was infected with the infection The inhibitory rate of HaCaT cells was proportional to its concentration, but when the dilution multiple of 1:100, the inhibitory rate of GoH3 was not significantly increased, and the average viral load of the 8 clinically isolated HPV 6/11 virus particles infected with HaCaT cells before adding GoH3 was (5.8 + 2.51) x 104copies/ml, and the average infection of HaCaT cells after the addition of GoH3 was observed. The viral load was (3.02 + 1.21) x 104copies/ml, and the average inhibition rate of GoH3 was (46.9 + 4.7)%.
Conclusion (1) PCR-RFLP method can be used for HPV DNA detection and typing, and integrin alpha 6 may play a role in the pathogenesis and recurrence of CA; (3) GoH3 has a certain inhibitory effect on HaCaT cells of HPV 6/11 infection.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R759

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