HPV基因分型与子宫颈鳞状细胞癌发生发展的分子病理机制初探

发布时间：2019-02-24 10:35

【摘要】：为探讨人乳头瘤病毒（Human papilloma virus，HPV）基因分型与子宫颈鳞状细胞癌发生发展的分子病理机制，本课题进行了以下研究：HPV基因分型在宫颈细胞学筛查中的应用及意义；子宫颈癌及其癌前病变中HPV感染及其基因型分布；HPV感染相关子宫颈癌和癌前病变中细胞周期蛋白的表达特征及规律；HPV16E6、E7癌基因在转录水平沉默的途径及沉默后细胞周期蛋白的表达变化。 收集2050例本院妇科门诊送检的宫颈脱落细胞，进行HPV基因分型检测；收集整理子宫颈癌及癌前病变和慢性宫颈炎石蜡组织标本，通过HE阅片筛选出129例标本入组，分为子宫颈鳞癌(squamouscarcinoma of the cervix, SCC)组、宫颈上皮内瘤变(cervical intraepithelialneoplasia, CIN)组、慢性宫颈炎组；应用PCR-反向点杂交法进行子宫颈癌与癌前病变HPV基因分型检测，检测数据用SPSS13.0软件进行统计分析；选取79例宫颈病变石蜡标本进行p16、p21、p53、Rb、CyclinD1等指标免疫组化（SP法）染色；采用免疫组化、RT-PCR、Western blot技术检测子宫颈癌HCC94、SiHa细胞中p16、p21、p53、Rb、CyclinD1基因及其蛋白表达，利用基因转染技术研究SiHa E6、E7基因瞬时沉默后细胞凋亡以及关键信号分子p16、p21、p53、Rb、CyclinD1、ki67的表达情况。 2050例宫颈上皮细胞标本中HPV感染阳性率为25.85%(530/2050)，最常见的类型依次为16、6、58、52、43型；109例子宫颈癌及其癌前病变标本中HPV阳性率为100％，最常见的基因型为HPV16型；随着病变程度的增加HPV单一感染比例逐渐升高，多重感染比例逐渐下降；p16蛋白在HPV(+)的子宫颈癌及其癌前病变中呈现过表达，在HPV(-)的正常组织中不表达；p53蛋白在正常宫颈组织中不表达，在SCC中偶见表达；Rb蛋白在正常宫颈鳞状上皮基底细胞中表达，，在CIN与SCC中表达缺失；CyclinD1蛋白在正常宫颈组织和CINⅠ中表达，部分CINⅡ、CINⅢ、SCC中表达缺失；在HPV16(+)SiHa细胞中，p16表达在mRNA和蛋白水平均高于HPV(-)的HCC94细胞，而CyclinD1表达在mRNA和蛋白水平均低于HCC94细胞；应用RNAi技术可以将SiHa细胞中HPV16E6、E7基因沉默，沉默效率可达70%以上，沉默后E6、E7mRNA和蛋白表达同步下降，细胞凋亡率上升，Ki67基因表达下降，p21基因表达上升。 宫颈细胞HPV基因分型对宫颈癌的早期筛查诊断具有重要意义；宫颈鳞癌及癌前病变发生与HPV感染直接相关，是高低危HPV亚型共同作用的结果，且高危型HPV持续感染是子宫颈癌及高级别癌前病变发生的必要条件；HPV16型病毒感染是国人子宫颈鳞癌形成的主要原因；p16、p21、p53、Rb、CyclinD1基因及蛋白的表达水平与HPV感染关系密切，其中子宫颈癌及其癌前病变中可见p16高表达及CyclinD1低表达，可作为辅助标记物用于子宫颈癌及癌前病变的病理诊断；靶向沉默HPV16E6、E7基因可有效抑制肿瘤细胞增殖并诱导凋亡。上述实验为深入探讨HPV基因分型与子宫颈癌发生发展的分子病理机制奠定了基础。
[Abstract]:In order to investigate the molecular pathological mechanism of human papillomavirus (Human papilloma virus,HPV) genotyping and cervical squamous cell carcinoma (SCC) occurrence and development, the following studies were carried out: the application and significance of HPV genotyping in cervical cytological screening; The distribution of HPV infection and its genotype in cervical carcinoma and its precancerous lesions, and the expression of cyclin in cervical cancer and precancerous lesions associated with HPV infection. The transcriptional silencing pathway of HPV16E6,E7 oncogene and the changes of cyclin expression after silencing. 2050 cases of cervical exfoliated cells were collected from gynecological outpatient department of our hospital for HPV genotyping. The paraffin tissue specimens of cervical carcinoma, precancerous lesion and chronic cervicitis were collected and sorted out. The specimens were selected by HE and divided into (squamouscarcinoma of the cervix, SCC) group and (cervical intraepithelialneoplasia, CIN) group, and the specimens were divided into two groups: cervical squamous cell carcinoma (squamouscarcinoma of the cervix, SCC) group, cervical intraepithelial neoplasia (cervical intraepithelialneoplasia, CIN) group, cervical squamous cell carcinoma (squamouscarcinoma of the cervix, SCC) group. Chronic cervicitis group; The HPV genotyping of cervical cancer and precancerous lesions was detected by PCR- reverse dot hybridization, and the data were analyzed by SPSS13.0 software. Seventy-nine paraffin specimens of cervical lesions were stained with SP method. Immunohistochemical and RT-PCR,Western blot techniques were used to detect the expression of p16 p21, p53, rbcyclin D1 gene and its protein in HCC94,SiHa cells of cervical cancer. SiHa E6 was detected by gene transfection technique. Apoptosis of E7 gene and expression of the key signal molecule p16, p21, p53, Cyclin D1 / ki67, after transient silencing of E7 gene. The positive rate of HPV infection in 2050 cases of cervical epithelial cells was 25.85% (530 / 2050), and the most common type was 166,58,52 / 43, and the positive rate of HPV was 100% in the specimens of cervical carcinoma and its precancerous lesions, the most common genotype was HPV16. With the increase of pathological degree, the proportion of single infection of HPV increased gradually, the proportion of multiple infection decreased gradually, p16 protein was overexpressed in cervical carcinoma of HPV () and its precancerous lesions, but not in normal tissues of HPV (-). P53 protein was not expressed in normal cervical tissues, but was occasionally expressed in SCC, Rb protein was expressed in basal cells of normal cervical squamous epithelium, and was absent in CIN and SCC. CyclinD1 protein was expressed in normal cervix and CIN 鈪

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