【摘要】：為探討人乳頭瘤病毒（Human papilloma virus，HPV）基因分型與子宮頸鱗狀細胞癌發(fā)生發(fā)展的分子病理機制，本課題進行了以下研究：HPV基因分型在宮頸細胞學(xué)篩查中的應(yīng)用及意義；子宮頸癌及其癌前病變中HPV感染及其基因型分布；HPV感染相關(guān)子宮頸癌和癌前病變中細胞周期蛋白的表達特征及規(guī)律；HPV16E6、E7癌基因在轉(zhuǎn)錄水平沉默的途徑及沉默后細胞周期蛋白的表達變化。 收集2050例本院婦科門診送檢的宮頸脫落細胞，進行HPV基因分型檢測；收集整理子宮頸癌及癌前病變和慢性宮頸炎石蠟組織標(biāo)本，通過HE閱片篩選出129例標(biāo)本入組，分為子宮頸鱗癌(squamouscarcinoma of the cervix, SCC)組、宮頸上皮內(nèi)瘤變(cervical intraepithelialneoplasia, CIN)組、慢性宮頸炎組；應(yīng)用PCR-反向點雜交法進行子宮頸癌與癌前病變HPV基因分型檢測，檢測數(shù)據(jù)用SPSS13.0軟件進行統(tǒng)計分析；選取79例宮頸病變石蠟標(biāo)本進行p16、p21、p53、Rb、CyclinD1等指標(biāo)免疫組化（SP法）染色；采用免疫組化、RT-PCR、Western blot技術(shù)檢測子宮頸癌HCC94、SiHa細胞中p16、p21、p53、Rb、CyclinD1基因及其蛋白表達，利用基因轉(zhuǎn)染技術(shù)研究SiHa E6、E7基因瞬時沉默后細胞凋亡以及關(guān)鍵信號分子p16、p21、p53、Rb、CyclinD1、ki67的表達情況。 2050例宮頸上皮細胞標(biāo)本中HPV感染陽性率為25.85%(530/2050)，最常見的類型依次為16、6、58、52、43型；109例子宮頸癌及其癌前病變標(biāo)本中HPV陽性率為100％，最常見的基因型為HPV16型；隨著病變程度的增加HPV單一感染比例逐漸升高，多重感染比例逐漸下降；p16蛋白在HPV(+)的子宮頸癌及其癌前病變中呈現(xiàn)過表達，在HPV(-)的正常組織中不表達；p53蛋白在正常宮頸組織中不表達，在SCC中偶見表達；Rb蛋白在正常宮頸鱗狀上皮基底細胞中表達，，在CIN與SCC中表達缺失；CyclinD1蛋白在正常宮頸組織和CINⅠ中表達，部分CINⅡ、CINⅢ、SCC中表達缺失；在HPV16(+)SiHa細胞中，p16表達在mRNA和蛋白水平均高于HPV(-)的HCC94細胞，而CyclinD1表達在mRNA和蛋白水平均低于HCC94細胞；應(yīng)用RNAi技術(shù)可以將SiHa細胞中HPV16E6、E7基因沉默，沉默效率可達70%以上，沉默后E6、E7mRNA和蛋白表達同步下降，細胞凋亡率上升，Ki67基因表達下降，p21基因表達上升。 宮頸細胞HPV基因分型對宮頸癌的早期篩查診斷具有重要意義；宮頸鱗癌及癌前病變發(fā)生與HPV感染直接相關(guān)，是高低危HPV亞型共同作用的結(jié)果，且高危型HPV持續(xù)感染是子宮頸癌及高級別癌前病變發(fā)生的必要條件；HPV16型病毒感染是國人子宮頸鱗癌形成的主要原因；p16、p21、p53、Rb、CyclinD1基因及蛋白的表達水平與HPV感染關(guān)系密切，其中子宮頸癌及其癌前病變中可見p16高表達及CyclinD1低表達，可作為輔助標(biāo)記物用于子宮頸癌及癌前病變的病理診斷；靶向沉默HPV16E6、E7基因可有效抑制腫瘤細胞增殖并誘導(dǎo)凋亡。上述實驗為深入探討HPV基因分型與子宮頸癌發(fā)生發(fā)展的分子病理機制奠定了基礎(chǔ)。
[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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