siRNA对宫颈癌细胞系 HPV16 E6基因的作用

目的 构建并筛选出最有效的HIV16 E6基因特异的小干扰RNA(amall interfering RNA，siRNA)表达载体，观察其对宫颈癌细胞中HIV16 E6基因表达的长期影响，探讨E6基因在宫颈癌发生过程中的分子作用机制，为临床HIV感染及宫颈癌治疗探索新方法。方法 构建Hairpin siRNA质粒，稳定转染宫颈癌SiHa细胞，鉴定转染细胞中的质粒DNA，通过Real-Time RT-PCR检测细胞中HPV16 E6mRNA表达，采用Westem-blot检测p53、p21等蛋白的变化。MTT法(四甲基偶氮唑盐微量酶反应比色法)检测SiHa细胞转染siRNA后细胞增殖曲线。结果 HIV16 E6A hairpin siRNA表达载体转染人宫颈癌SiHa细胞，可以在细胞内长期表达siRNA，有效抑制细胞内HIV16 E6基因的表达。E6A siRNA能抑制细胞生长，作用持续达4个月以上。结论 利用siRNA表达载体抑制整合在细胞中的外源HIV E6病毒癌基因可能是治疗HIV感染和宫颈癌的一种新的理想方法。     

人乳头状瘤病毒16型E6、E7基因转染的人宫颈上皮永生化细胞系的建立及鉴定

目的 建立HPV16 E6、E7基因转染的胎儿宫颈上皮永生化细胞系，为体外研究宫颈上皮HPV感染及其癌变机制提供模型。方法 探索胎儿宫颈上皮细胞原代培养的方法，用逆转录病毒法将HPVl6E6、E7基因转染原代培养的胎儿宫颈上皮细胞，筛选出稳定表达的细胞系，进行传代培养；应用RT-PCR检测细胞中HPV16E6、E7基因的表达，用绘制细胞生长曲线、形态学观察、软琼脂集落形成实验、流式细胞术、Transwell Insert体外侵袭实验和裸鼠成瘤实验等方法对永生化的胎儿宫颈上皮细胞进行鉴定。结果 采用组织块培养法。选用Ker-SFM无血清培养基培养出纯度和生长状态良好的胎儿宫颈上皮原代细胞，可以利用高滴度的逆转录病毒作为载体，感染原代细胞，成功地建立一株永生化的胎儿宫颈上皮细胞系。永生化的细胞仍呈多边形，角蛋白阳性，仍维持上皮细胞表型，雌、孕激素受体阴性，染色体核型发生明显变化，但目前尚无软琼脂集落形成能力、不能穿过Matrigel基底膜，不能在裸鼠体内形成肿瘤。结论 转染HPV16E6、E7基因能使胎儿宫颈上皮细胞永生化，永生化细胞仍维持上皮细胞表型，染色体核型发生明显变化，但尚未完全恶性转化。     

雌激素对宫颈癌细胞生长及人乳头瘤病毒18 E7基因表达的调节

目的　研究雌激素 (1 7β -estradiol,E2 )对宫颈癌HeLa细胞生长的影响及细胞中人乳头瘤病毒(HumanPapillomavirus,HPV) 1 8E7病毒癌基因表达的调节作用。方法　用细胞计数法、流式细胞术检测不同浓度E2 对细胞生长及增殖的影响 ;用RT -PCR方法半定量检测E2 对细胞中HPV1 8E7mRNA表达的影响。结果　①浓度≤ 1 0 μM时 ,E2 对HeLa细胞的生长起促进作用 ,有剂量、时间依赖性 (均P =0 0 0 0 ) ,0 5 μM的E2 作用 5d时对HeLa细胞生长的促进作用最强 ;浓度≥ 5 0 μM时 ,E2 对HeLa细胞生长有明显的抑制作用 (均P =0 0 0 0 )。②与对照组相比 ,浓度≤ 1 0 μM时 ,E2 均使HeLa细胞S期比例增加 ,当浓度为 0 5 μM时E2 使S期细胞比例增加最明显 (P =0 0 1 0 )。③ 0 5、 1 0 μM的E2 使HeLa细胞中HPV1 8E7mRNA表达明显增加 (均P =0 0 0 0 ) ;1 0 μM的E2 对细胞中HPV1 8E7mRNA表达的促进作用较 0 5 μM的E2 更强 (P =0 0 0 0 )。结论　①低浓度的E2 (≤ 1 0 μM)促进HeLa细胞生长 ,使S期细胞比例增加 ;高浓度E2 (≥ 5 0 μM)明显抑制HeLa细胞的生长。②E2 能促进HeLa细胞中HPV1 8E7mRNA的表达     

可遗传性表达人乳头瘤病毒18型E6发夹状RNA干扰质粒对宫颈癌细胞生长特性的影响

目的:探讨稳定转染可遗传性表达HPV18E6shRNA质粒对HPV18阳性的宫颈癌HeLa细胞株生长特性的影响。方法:实验组为稳定转染表达HPV18E6shRNA的pE6-1shRNA质粒和含E6基因突变点的pE6-2shRNA质粒的宫颈癌Hela细胞株,对照组为稳定转染pSUPER空质粒和未转染质粒的宫颈癌HeLa细胞株。MTS测各组细胞的生长曲线,流式细胞仪检测各组细胞周期的变化和增殖指数;软琼脂细胞培养分析各组细胞集落形成能力。结果:MTS显示pE6-1shRNA组较pE6-2shRNA、空质粒pSUPER组及未转染组的HeLa细胞增殖率低,差异有显著性(P<0.05);流式细胞仪检测,pE6-1shRNA组G0-G1期细胞数较pE6-2shRNA组增加了12.5%,较未转染的HeLa细胞组增加了13·3%(均P<0.05);pE6-1shRNA组进入S期的细胞数较pE6-2shRNA组减少了9·4%,较未转染的HeLa细胞组减少了8.5%(均P<0.05),G2-M期细胞数在各组差异无显著性(P>0.05)。转染pE6-1shRNA组细胞增殖指数23.7%;而pE6-2shRNA组细胞增殖指数36.1%,未转染组细胞增殖指数36.9%。细胞软琼脂培养2周,pE6-1shRNA组的HeLa细胞的克隆形成率4.2%,较未转染组的HeLa细胞克隆形成率7.2%低(P<0·05,χ2=5.56),也低于转染pE6-2shRNA和pSUPER组(P>0.05)。结论:稳定转染可遗传性表达HPV18E6shRNA质粒的宫颈癌HeLa细胞,其细胞增殖周期和细胞生长受到抑制。     

Human papillomavirus type 16 E6 and E7 gene variations in Indian cervical cancer

OBJECTIVES: Human papillomavirus type 16 is a causative factor for development of cervical cancer. The E6 and E7 genes of HPV 16 are critical to the process of immortalization and transformation of host cells. Recent reports suggest that variants of these two genes may contribute to the risk of malignant progression of cancer in the uterine cervix. However, no data exist on sequence variations of HPV 16 E6 and E7 genes that may exist in India. Therefore, we examined intratype variations in the E6 and E7 viral genes in DNA isolated from HPV 16-positive cervical scrapes and biopsies. METHODS: The open reading frames of the E6 and E7 genes were amplified by PCR and then directly sequenced by the fluorescent dye dideoxy termination method.Results. In addition to the prototype E6 gene sequence, five sets of mutations of the E6 gene were identified. The European prototype (350T) was detected in 9.1% of the study group while the European variant (350G) was seen in 28% of patients. The remaining variants (a combination of the 350G mutation with 335T, 145T, or 419G) were significantly associated with cases compared to controls. The 350G + 145T variant was found at much higher incidence in cases in younger women, suggesting that this variant may be associated with aggressive tumor behavior. Interestingly the 350G + 419G combination was found only in controls. There was no significant association between the four genotypes of E7 and any stage of tumor progression or age. CONCLUSIONS: The results indicate that specific mutations in the E6 gene are found in young Indian women with high-grade squamous intraepithelial lesions and invasive cancer, suggesting that these mutations represent more oncogenically active HPV 16. Whether this increased oncogenecity is due to differences in p53 inactivation, ineffective keratinocyte differentiation, and/or altered response to the immune system by these oncogenic E6 mutants remains to be clarified.     

人乳头瘤病毒基因亚型与宫颈病变的关系

目的 研究不同程度的子宫颈癌前病变者感染HPV的基因亚型，以探讨不同基因型HPV感染的致癌性。方法 研究对象系3．56例宫颈HPV感染的患者。以第二代杂交捕获（HC1I）试验检测13种HPVH，同时采用核酸分子快速杂交基因分型技术进行21种HPV-DNA亚型分析；所有研究对象均行宫颈薄层液基细胞学检测（LCT）及阴道镜下宫颈多点活检。根据细胞与病理学诊断有无宫颈病变及其病变程度分组。①宫颈癌与高度鳞状上皮内瘤变（HSIL）组：包括宫颈浸润癌9例、CINⅢ57例、CINⅡ53例；②低度鳞状上皮内瘤变（LSIL）组：包括CINⅠ112例、HPV感染52例；③正常或炎症组：宫颈细胞与组织学正常或炎症者73例。通过HPV-DNA亚型分布，分析HPV感染基因型与宫颈病变程度的关系。结果 HPV-H与宫颈癌变密切相关，本组高度病变以上患者中HPV-H阳性率达98．4％；随着宫颈病变程度加重，HPV-H双重与多重感染有增加趋势，在正常与炎症组、LSIL组与HSIL组中两种以上HPV-H基因型感染者分别占13．7％、17．7％和36．6％，HSIL组明显高于其他两组，差异有显著性（P〈0．01）；本组HPV亚型感染频度由高到低依次为HPV-16（32．9％）、58（18．8％）、52（16．9％）、18（9．0％）、33（8．2％）、68（7．3％）、11（低危型，6．5％）、31（6．2％）、53（5．6％）、39（5．3％）、CP8304（5．1％）、66（3．9％）、6（2．5％）和51、56、45、35、59、43、44、82等少见型；HPV-16、58、33、52与18型在高度鳞状上皮内瘤样病变（HSIL）中感染率分别为56．4％、19．3％、16．0％、12．6％和8．4％，说明这些亚型致癌性更强；两种HPV-DNA检测技术所获结果，具有很高的一致性。结论本组宫颈病变患者感染HPV型别以16、58、52、18、33较为多见，HPV-16、58、33、52与18型致癌性较强；HPV-DNA分型检测在临床诊断与判断预后上具有重要意义。     

Polymorphism in the E6 gene of human papillomavirus type 16 in the cervical tissues of Korean women

The aim of this study was to identify sequence variants in the HPV 16 E6 gene in Korean women and to examine the possible association between these sequence variants and cervical cancer development. We examined the HPV 16 DNA of 215 patients with no cervical disease (NCD) (n = 105) or with cervical neoplasia (n = 110) [cervical intraepithelial neoplasia (CIN), n = 61; invasive cervical carcinoma (ICC), n = 49] using the nested polymerase chain reaction (PCR) and PCR-directed sequencing methods. Fifty-four (NCD, n = 10; CIN, n = 17; ICC, n = 27) of the 215 samples contained HPV 16 E6 DNA, but only two (7.4%) of 27 ICC samples had prototype sequences. The most frequently found variation was D25E (in NCD, n = 8, 80%; in CIN, n = 9, 52.9%; in ICC, n = 23, 85.2%). This is a rare variation in western countries. No significance difference was found between the frequencies of D25E variation in cancerous and non-cancerous lesions. Among the 11 kinds of variants identified, four variants were novel and have been registered with GenBank. This study demonstrates that the D25 variant is the most prevalent E6 genomic variant type in Korean population. However, it was not found to be associated with an increased risk of ICC.     

Molecular variants of human papillomavirus type 16 and risk for cervical neoplasia in South Africa

Non-European variants of human papillomavirus (HPV) type 16 are generally associated with a greater risk of cervical neoplasia than European prototype variants. We investigated whether this association would persist in a population in which non-European HPV 16 variants were more common. We sequenced HPV 16 isolates in cervical samples collected from 93 Black South African women enrolled in a cervical cancer screening study and examined associations between cervical neoplasia identified though colposcopy with cervical biopsy and the specific HPV 16 variant identified. The European prototype variant (EP) was the most commonly identified variant in this population (47% of all isolates), but African variants (Af-1 and Af-2) were also quite common (41% of all isolates). In contrast to previous studies, we found no evidence that non-European variants were associated with an increased risk of neoplasia. Rather, most of the HPV 16-associated cancers were found in association with EP (71% of 14 cases). In this setting where African HPV 16 variants were common, no increased risk for cervical neoplasia was found among women with these variants compared with other HPV 16 variants.     

反义HPV16 E6/E7-EGFP重组质粒的构建及其对宫颈癌SiHa细胞的促凋亡作用

目的:构建HPV16早期基因E6/E7的反义重组质粒,探讨其对SiHa细胞的促凋亡作用。方法:将HPV16E6/E7基因片段反向克隆于真核表达载体pEGFP-C1并转染SiHa细胞,用RT-PCR方法检测转染后SiHa细胞E6、E7基因mRNA的表达,West-ernblot方法检测转染后E6/E7蛋白的表达,流式细胞仪检测转染后细胞的凋亡率。结果:成功构建携带HPV16E6/E7基因反义片段的真核表达载体,转染该质粒后,SiHa细胞E6、E7基因的mRNA和蛋白均明显下调;转染后细胞凋亡率为(59.3±11.3)%,明显高于转染空载体组[(9.4±1.8)%]和未转染组[(2.1±0.4)%](P<0.05)。结论:反义HPV16E6/E7基因可下调宫颈癌细胞中E6/E7癌基因的表达,诱导宫颈癌细胞凋亡,为宫颈癌的基因治疗提供了实验依据。     

A study of women's knowledge regarding human papillomavirus infection, cervical cancer and human papillomavirus vaccines

AIMS: Human papillomavirus (HPV) infection is a common sexually transmitted viral infection and is associated with the development of cervical cancer. HPV vaccines are now undergoing phase 3 clinical trials in Australia. It is likely that an HPV vaccine will become licensed for use in the near future. METHODS: Ninety women aged 18-30 years from three different groups (those attending a dysplasia clinic, a local university health service and participants currently involved in a phase 3 HPV vaccine trial) completed a questionnaire assessing their knowledge base regarding HPV infection, cervical cancer, Pap tests and HPV vaccines. RESULTS: Respondents demonstrated good understanding of the Pap test and interpretation of an abnormal result. Most respondents (89%) had heard of HPV and attributed a number of different clinical symptoms to infection. For women who had not heard of an HPV vaccine, 79% of respondents stated that the most common resource they would use to obtain further information is their general practitioner. DISCUSSION: Many women do not understand the risk factors for HPV infection, the clinical problems it may cause and the potential long-term complications of infection. Few women have heard of a HPV vaccine, but most women surveyed would approach their general practitioner for more information if one became available. CONCLUSION: This study highlights the need for further education regarding HPV infection and the potential long-term complications such as cervical cancer. It also demonstrates that education of general practitioners regarding an HPV vaccine is essential, as this is the most likely resource women will use to obtain further information in the future.     

人乳头瘤病毒亚型与宫颈上皮内瘤变的相关性

目的探讨人乳头瘤病毒(HPV)各亚型感染或多亚型感染与宫颈上皮内瘤变(CIN)的相关性。方法 2007年11月至2008年4月在天津医科大学总医院采用PCR扩增、基因芯片探针杂交分型检测法检测98例CIN患者的HPV亚型。以35岁为界将98例CIN患者分为两组,年龄≤35岁者为A组(43例);年龄>35岁者为B组(55例)。结果 98例CIN中HPV阳性率,CINⅠ为72.41%(21/29),CINⅡ为75%(27/36),CINⅢ为96.97%(32/33)。HPV单重感染率在CINⅠ、CINⅡ和CINⅢ中分别为41.38%(12/29),44.44%(16/36)和57.58%(19/33);HPV双重感染率在CINⅠ、CINⅡ和CINⅢ中分别为27.59%(8/29),19.44%(7/36)和27.27%(9/33);HPV多重感染率在CINⅠ、CINⅡ和CINⅢ中分别为3.45%(1/29),11.11%(4/36)和12.12%(4/33)。CINⅠ中主要为低危型HPV感染,CINⅢ中以HPV16为主导感染亚型。AB两组HPV16感染率分别为20.93%(9/43),40%(22/55),差异具...     

人乳头状瘤病毒感染的宫颈上皮内肿瘤和宫颈鳞状细胞癌中的间期细胞遗传学分析

目的探讨人乳头状瘤病毒（HPV）感染的宫颈上皮内肿瘤（CIN）和宫颈鳞状细胞癌中的间期细胞遗传学改变。方法对HPV感染的9例CIN（其中CINⅠ2例、CINⅡ3例、CINⅢ4例）和4例宫颈鳞状细胞癌的石蜡包埋组织，应用组织切片间期细胞核原位杂交技术检测染色体11、16、17和X的数目异常，3例正常宫颈鳞状上皮作为对照。结果除2例CINⅠ的患者中被检染色体无异常外，其余11例（包括3例CINⅡ，4例CINⅢ以及4例宫颈浸润性鳞状细胞癌）均出现1条或多条被检测染色体的异常，其中4例分别发现了染色体11、17和X单体型。结论HPV16感染与11、16、17及X号染色体数目异常有关，而HPV DNA与宿主细胞染色体整合可能导致染色体的单体型出现。     

HPV 16 DNA in autopsy material of a metastatic cervical carcinoma

Summary Human papillomavirus (HPV) DNA has been regularly detected in primary cervical carcinomas and in some metastatic lesions. Using Southern blot hybridization on autopsy material we found HPV 16 DNA in a primary cervical carcinoma and in multiple metastases therefrom.     

5-hmC在宫颈上皮内瘤变病理诊断中的意义及其与HPV感染的相关性#br#

目的探讨5-羟甲基胞嘧啶(5-hmC)在宫颈上皮内瘤变和宫颈鳞癌中的表达,高危型HPV在上述病变中的感染率,分析两者之间的相关性。方法应用免疫组织化学技术分别检测正常组(31例)、LSIL(42例)、HSIL(61例)和宫颈鳞癌(91例)5-hm C的表达和高危型HPV感染率。结果5-hm C阳性率在正常组、LSIL、HSIL以及宫颈鳞癌组逐渐降低,差异有统计学意义(χ²=21.146,P=0.000);高危型HPV感染率随着病变的加重逐渐增高,差异有统计学意义(χ²=69.964,P=0.000);5-hm C和高危型HPV感染率都与宫颈上皮内瘤变的组织学分级具有相关性(r=-0.512,P=0.000)、(r=0.541,P=0.000);且两者之间呈负相关(r=-0.315,P=0.000)。结论5-hm C的表达水平对宫颈上皮内瘤变分级诊断具有鉴别意义并且与高危型HPV感染密切相关,两者可能共同促进了宫颈上皮内瘤变向宫颈鳞癌的转化。     

Association of human leukocyte antigen and T cell message with human papillomavirus 16–positive cervical neoplasia in Japanese women

To investigate whether an association exists between human leukocyte antigen (HLA) haplotype and cervical neoplasia within the Japanese population, we analyzed the human papillomavirus (HPV) genotypes, the HLA class I specificities and class II alleles, and the T-cell responses in the lesions of patients with cervical neoplasia. Eighty-one patients, consisting of 62 cervical intraepithelial neoplasia (CIN) lesions and 19 invasive cervical cancers (ICC), were examined. The frequencies of HPV infection in the CIN I/II and CIN III/ICC groups were 68.0% (17/25) and 80.4% (45/56), respectively. All patients and 138 local Japanese controls were analyzed for HLA-A, HLA-B, HLA-DRB1, and HLA-DQB1. For major histocompatibility complex (MHC) class II HLA-DRB1 alleles, the frequency of DRB1*0901 was significantly elevated in HPV 16-positive CIN III/ICC patients compared with controls (59.3% versus 29.7%, P = 0.0031, OR = 3.44). Similarly for the HLA-DQB1 alleles, a significant increase in the DQB1*03032 frequency was observed in HPV 16-positive CIN III/ICC patients compared with controls (59.3% versus 28.3%, P = 0.0018, OR = 3.69). In the analysis of the T-cell responses in the lesions, Fas ligand was detected at a decreased frequency in HPV 16-positive CIN III/ICC patients with the HLA-DRB1*0901-DQB1*03032 haplotype. The presence of helper T cell-specific messenger RNAs in the cervical lesions supports an association among MHC class II, helper T cells, the immune response to HPV, and the development of cervical carcinoma. Accordingly, a specific MHC class II haplotype, DRB1*0901-DQB1*03032, may be a risk factor for cervical carcinoma in the Japanese population.     

慢病毒介导短发夹状RNA沉默生存素基因对人异位内膜细胞增殖和凋亡的影响

目的:研究慢病毒介导生存素(survivin)基因特异性短发夹状RNA(shR-NA)对人异位内膜细胞中survivin基因表达及细胞增殖和凋亡的影响。方法:构建靶向survivin基因的shRNA慢病毒表达载体,将重组慢病毒感染原代培养的异位内膜细胞,以未转染及空载慢病毒感染异位内膜细胞为对照。采用RT-PCR、Western blot法分别检测各组异位内膜细胞中survivin mRNA和蛋白表达的变化,用四甲基偶氮唑蓝法检测细胞的增殖,Cy5-Annexin V/PI双染标记流式细胞仪测定细胞凋亡。结果:与未转染细胞及空载慢病毒转染细胞相比,shRNA慢病毒感染人异位内膜细胞后survivin mRNA及蛋白表达水平明显下降,survivin mRNA及蛋白表达抑制率分别为64%和59%。转染后1～5天实验组细胞生长速度较阴性对照组及空白对照组明显减慢,除第1天外,差异均有统计学意义(P0.01);阴性对照组及空白对照组细胞生长速度的差异无统计学意义(P0.05)。流式细胞仪分析显示,实验组细胞凋亡率为(41.61±3.64)%,明显高于阴性对照组的(9.14±1.88)%和空白对照组的(7.07±1.16)%(P0.01),后两组的差异无统计学意义(P0.05)。结论:慢病毒介导survivin基因特异性shRNA可有效沉默异位内膜细胞中survivin基因表达,明显抑制异位内膜细胞的增殖并促进细胞凋亡。