【作者】 张小燕；

【导师】 卞美璐； 陈庆云；

【作者基本信息】 中国协和医科大学 ， 妇产科学， 2007， 博士

【摘要】 目的:1.评价杂交捕获二代和流式荧光杂交技术在子宫颈人乳头状瘤病毒(HumanPapillomavirus,HPV)感染检测及宫颈病变诊治中的应用价值。2.探讨中药保妇康栓及主要成分莪术油对HPV16的抑制作用。方法:1.选择在中日友好医院妇产科宫颈病变诊治中心行宫颈癌筛查的细胞学诊断异常的患者(≥ASC-US)872例,采用杂交捕获二代(HC-Ⅱ)进行13种高危型HPV的检测,同时对组织病理学≥CIN2者采用流式荧光杂交HPV分型检测法进行15种HPV亚型分型检测,比较不同方法检测结果的一致性;按宫颈组织病理学结果分为NILM组、宫颈上皮内瘤变(CIN)Ⅰ、Ⅱ、Ⅲ、宫颈癌组。以组织病理学诊断为金标准,评价高危型HPVDNA检测在宫颈病变诊断中的应用价值。采用Spearman等级相关分析HPV病毒载量与宫颈病变级别的关系。计算各HPV亚型在不同病理级别组中的阳性率。2.以含保妇康栓的培养基体外培养宫颈癌细胞系CaSki和宫颈永生化细胞系H8,以含莪术油的培养基体外培养宫颈癌细胞系SiHa、CaSki和H8,观察药物作用后细胞形态学变化;采用四甲基偶氮唑蓝比色法(MTT)、流式细胞术检测不同浓度药物对细胞增殖的影响;应用逆转录-聚合酶链反应技术(ReverseTranscriptase Polymerase Chain Reaction,RT-PCR)检测人乳头状瘤病毒HPV16亚型E6E7mRNA的表达。结果:1.HC-Ⅱ法诊断宫颈高度病变(≥CIN2)的灵敏性、特异性、阳性预告值、阴性预告值分别为93.1%、66.8%、59.9%、94.8%。HC-Ⅱ阳性的ASC-US和ASC-H患者中宫颈高度病变的检出率为28.9%、30.0%,阴性者中检出率为2.3%.3.7%(P＞0.05)。NILM组HPV病毒载量与CIN以及宫颈癌组有显著性差异(P＜0.05),CIN以及宫颈癌组HPV病毒载量无显著性差别(P＞0.05)。流式荧光杂交与HC-Ⅱ两种方法对高危型HPV感染的检出结果总符合率92.4%,kappa值0.623。流式荧光杂交HPV检测结果显示≥CIN2患者最常见的基因型为16、18、52、58、31。HPV16阳性率随病变级别的增加迅速上升。CINⅡ组常见HPV亚型16、52、11、18、58、56/66,CINⅢ组常见HPV亚型16、58、18、52、31,宫颈癌组常见亚型16、18、11、31、52、58。2.不同浓度的保妇康栓可以抑制宫颈癌细胞系Caski和宫颈永生化细胞H8的增殖;在保妇康栓作用下,Caski细胞G1期减少(P＜0.05),G2、S期增加(P＜0.05),Caski细胞凋亡率高于对照组(P＜0.05),而对H8细胞周期和凋亡率影响不明显;两种细胞系HPV16E6E7基因片段mRNA表达均明显低于对照组(P＜0.01),无剂量依赖关系。3.不同浓度的莪术油可以抑制细胞的增殖,莪术油作用于SiHa细胞,G1期细胞减少(P＜0.05),G2、S期细胞增加(P＜0.01),细胞阻滞于G2、S期。莪术油作用于CaSki、H8细胞,G1期细胞减少(P＜0.01),S期细胞增加(P＜0.01),使细胞阻滞于S期。加药组SiHa、CaSki和H8细胞凋亡率高于对照组,有统计学差异(P＜0.01,P＜0.01,P＜0.05);三种细胞HPV16E6E7基因片段mRNA表达均明显低于对照组(P＜0.01)。结论:1.随着宫颈细胞学和组织病理学级别的升高,高危型HPVDNA的感染率呈趋势性升高。高危型HR-HPVDNA检测对于ASC-US有分流作用,对于ASC-H的分流作用有待进一步探讨。HC-Ⅱ法检测HR-HPV病毒载量与CIN以及宫颈癌发生有关,但与宫颈病变的严重程度无关。流式荧光杂交技术与HC-Ⅱ检测结果一致性好,是有效的HPV分型检测方法。HPV16、18、52、58、31是≥CIN2患者最常见的5种HPV感染亚型。2.保妇康栓及其主要成分莪术油可抑制宫颈癌细胞系SiHa、CaSki以及宫颈永生化细胞系H8增殖,其机制可能与抑制HPV16E6E7表达有关。更多还原

【Abstract】 Objectives1.To investigate the applicable value of Hybrid CaptureⅡ(HC-Ⅱ) and the flow fluorescent hybridization assay on human papillomavirus(HPV) detecting.To evaluate the clinical accuracy of the two method for the diagnosis of cervical neoplasia.To detect the most common HPV subtype in different grades of cervical lesions.2.To investigate the mechanism of the proliferative inhibition of HPV16 by curcuma aramatica oil.Methods1.872 patients with abnormal cervical cytology results who accepted cervical cancer screening in China-Japan Friendship Hospital from June 2006 to April 2007 took HR-HPV testing(HC-Ⅱmethod) and all patients underwent colposcopy and biopsy..All patients were divided into five groups as NILM,CINⅠ,CINⅡCINⅢand invasive cervical cancer(ICC).Association between HR-HPV load and CINs were evaluated by Spearman rank correlation.Those with histological pathology diagnosis as cervical intraepithelial neoplasia(CIN)Ⅱor above were HPV subtyped by flow fluorescent hybridization assay.The accordance rate of the two assays was compared. The applicable value on diagnosis of cervical lesions by the two methods was evaluated according to the diagnosis of histological pathology.The infection rates of different genotypes in each group were calculated.2.The human cervical cancer cell line-CaSki and the immortalized cervical epithelial cell-H8 were cultured with different concentration of baofukang suppository The human cervical cancer cell line-CaSki、SiHa and the immortalized cervical epithelial cell-H8 were cultured with different concentration of curcuma aramatica oil.The proliferative inhibition of different concentration of curcuma aramatica oil or baofukang suppository on cell lines was measured by methyl thiazolyl tetrazolium(MTT) assay and flow cytometry assay;The mRNA expression of HPV16 E6E7 was determined by RT-PCR semi-quantitatively.Results 1.The sensitivity,specificity,positive predictable value and negative predictable value for CIN2 by HC-Ⅱwere 93.1%、66.8%、59.9%、94.8%,respectively.There was significant differentce between NILM and CINs or ICC,while no significant difference was observed among CINⅠ,CINⅡ,CINⅢand ICC.The total accordance rate of the flow fluorescent hybridization assay and HC-Ⅱwas 92.4%.The most common HR-HPV subtypes among patients with CIN2 or above were HPV16,18,52,58, 31.The positive rate of HPV 16 increased rapidly with the severity of the disease.The most common HPV types were 16,52,11,18,58 in CINⅡ,group,16,58,18,52,31 in CINⅢgroupand 16,18,31,52,58 in ICC group.2.The growth of Caski and H8 was inhibited by different concentration of Baofukang suppository.After Baofukang Suppository treatment,Caski and H8 cell line showed decreased levels of HPV16 E6E7 mRNA than that in the control group.The growth of CaSki,SiHa and H8 was inhibited by different concentration of curcuma aramatica oil.SiHa cells were accumulated in G2、S phase(P＜0.01),while CaSki and H8 cells were accumulated in S phase(P＜0.01).The apoptosis rate in CaSki,SiHa and H8 cells was higher than that in control group(P＜0.01, P＜0.01,P＜0.05).CaSki,SiHa and H8 cell showed decreased level of HPV16 E6E7 mRNA than that in the control group after curcuma aramatica oil treatment(P＜0.01).Conclusions1.The positive rate of HR-HPV increases with the severity of the cervical lesions.HR-HPV testing is useful in triage of ASC-US,while its triage role for ASC-H need further study.The HR-HPV load is significantly associated with the presence of CINs and ICC.HPV viral load does not increase with severity of cervical neoplasia.The 5 most common subtypes in patients≥CIN2 or above are HPV16,18,52,58,31.2.The results suggest that Baofukang Suppository and curcuma aramatica oil may execute its antiHPV activity primarily by decreasing the expression of HPV 16E6E7 in cervical cancer cell line and immortalized cervical epithelial cell.更多还原