靶向切割HPV16E6 mRNA的核酶诱导宫颈癌细胞CaSKi凋亡的研究(英)

背景与目的:人乳头瘤病毒与宫颈癌的发生相关,核酶是一种能切割靶RNA的特殊RNA。本研究旨在探讨转染了靶向切割HPV16E6mRNA核酶的宫颈癌细胞株的表型,以及核酶对宫颈癌细胞增殖与调亡的作用。方法:计算机设计特异性切割HPV16E6mRNA的核酶,构建抗HPV16E6核酶(HRz)的真核表达质粒。以脂质体法将抗HPV16E6核酶、空载体质粒分别导入CaSKi细胞,命名为CaSKi-R和CaSKi-P细胞。Northern杂交检测3种细胞中E6基因的表达。流式细胞仪检测3种细胞的凋亡率,将3种细胞在相差显微镜和荧光显微镜下观察,采用荧光(Hoechst33342)染色和TUNEL(TDT-mediateddUTPnickendlabeling)两种方法测定细胞凋亡。流式细胞术测定HPV16E6、c-myc、bcl-2、p53、Fas等蛋白的表达。结果:RNA点杂交证实核酶mRNA能稳定表达于CaSKi-R细胞中。Northern杂交证实CaSKi-R中E6mRNA表达水平较CaSKi细胞明显降低,而CaSKi-P和CaSKi细胞的E6mRNA表达水平无差异。CaSKi-R细胞凋亡率明显高于CaSKi和CaSKi-P细胞,细胞周期阻滞于G2期,S期细胞百分率下降。抗HPV16E6核酶能明显减少CaSKi-R细胞中HPV16E6、c-myc、bcl-2蛋白的表达,增高p53蛋白的表达;这种现象并不发生于CaSKi-P细胞中。Fas蛋白的表达在3种细胞中都相近。结论:抗HPV16E6核酶的导入能诱导宫颈癌细     

抗HPV16E6核酶对宫颈癌细胞恶性表型的影响

目的：研究特异性抗HPV16E6核酶对宫颈癌细胞恶性表型的影响。方法：以脂质体法将抗HPV16E6核酶、空载体质粒分别导入CaSKi细胞，命名为CaSKiR， CaSKiP细胞。测定CaSKi，CaSKiR，CaSKiP 3种细胞的生长曲线和软琼脂克隆形成率，流式细胞仪检测3种细胞中HPV16E6, PCNA, CerbB2蛋白的表达。将裸鼠分为3组，分别在皮下接种CaSKi，CaSKiR，CaSKiP细胞，检测细胞在裸鼠体内的成瘤能力；另取一组裸鼠，每只在右侧接种CaSKi细胞，左侧接种CaSKiR细胞，对比这两种细胞的致瘤性。分析特异性抗HPV16E6核酶对宫颈癌细胞恶性表型的影响。结果：CaSKiP，CaSKi细胞生长速率相近, CaSKiR细胞的生长速度明显降低。CaSKiR细胞的软琼脂克隆形成率明显低于CaSKi和CaSKiP细胞。与CaSKi细胞相比，CaSKiR细胞表达HPV16E6，PCNA，CerbB2蛋白明显减少，而CaSKiP细胞无此改变。CaSKi和CaSKiP在裸鼠体内的致瘤性无显著差异，而CaSKiR的成瘤性显著低于CaSKi。结论：抗HPV16E6核酶的导入能部分逆转宫颈癌CaSKi细胞株的恶性表型，其原因可能在于病毒癌基因E6表达的降低，以及由此而引起的PCNA，CerbB2基因表达的降低。     

抗HPV16E6核酶增加宫颈癌CaSKi细胞对顺铂敏感性的研究

目的：研究抗HPV16E6核酶（Ribozyme)导入宫颈癌CaSKi细胞对顺铂（DDP）敏感性的影响。方法：以脂质体法将抗HPV16E6－Ribozyme,空载体质粒分别导入CaSKi细胞,命名为CaSKi-R,CaSKi-P细胞,点穴交检测核酶在细胞中的表达,Northern杂交检测3种细胞中E6基因的表达,检测3种细胞的细胞生长曲线和克隆形成实验,MTT法检测3种细胞对DDP的敏感性,Annexine/PI双标法检测细胞凋亡率,流式细胞术检测Bcl-2,P53,Bax蛋白表达,结果：点杂交证实核酶能在CaS-Ki-R细胞中稳定表达,Northern杂交证实CaSKi-R中表达E6较CaSKi-P,CaSKi明显降低,与CaSKi-P和CaSKi细胞比较,CaSKi-R细胞的生长速率明显减慢和克隆形成率下降,对DDP的敏感性明显增加（P＜0．01）,凋亡率明显增加（P＜0．01）P53,Bax蛋白表达明显增加,Bcl-2蛋白表达明显减少（P＜0．01）,CaSKi-P和CaSKi细胞比较无差异（P＞0．05）,结论：抗HPV16E6－Ribozyme抑制了CaSKi-R细胞的生长和克隆形成率,CaSKi-R细胞对顺铂的敏感性增加。     

特异性核酶增强宫颈癌细胞对多种化疗药物的敏感度研究

目的研究抗HPV16E6核酶（Ribozyme）在宫颈癌CaSKi细胞对多种化疗药物在体内外敏感度的影响。方法以脂质体法将抗HPV16E6-Ribozyme、空载体质粒分别导入CaSKi细胞,命名为CaSKi、CaSKi-R、CaSKi-P细胞。MTT敏感实验检测多种化疗药物对三种细胞的抑制率;建立三种宫颈癌细胞移植瘤裸鼠模型,检测顺铂(DDP)对其抑制作用;透射电镜观察顺铂作用后的三种细胞形态。结果与CaSKi-P、CaSKi细胞比较,DDP、VCR、5-Fu、MMC对 CaSKi-R细胞的抑制率明显增加（P<005）,CaSKi-R细胞对 DDP、VCR、5-Fu、MMC的敏感度明显增加; ADM、MTX、INF、Taxol、Ara-C、CTX对 CaSKi-R细胞的抑制率无明显变化（P>0.05）,敏感度无明显变化;顺铂作用后CaSKi-R、CaSKi-P和CaSKi细胞裸鼠移植瘤的重量分别为（0.09±0.03）g、（0.26±0.07）g和（0.26±0.05）g（P＜005）,抑制率分别为81.63%、62.32%和63.38%;顺铂作用后CaSKi-R细胞出现明显的凋亡改变, 而CaSKi、CaSKi-P细胞不明显。结论抗HPV16E6-Ribozyme增加了CaSKi细胞对DDP、VCR、5-Fu、MMC的敏感度,抑制了宫颈癌细胞裸鼠移植瘤的生长并增加对DDP的敏感度。     

抗HPV16核酶对宫颈癌CaSKi细胞放疗敏感性的影响

背景与目的：人乳头瘤病毒（human papillomavirus,HPV)是宫颈癌最主要的致病因素,E6是主要的致癌基因之一;高危HPV基因型,如HPV16的E6蛋白表达水平是维持宫颈癌恶性表型的必要条件。而放射治疗是目前宫颈癌治疗的标准和有效的方法。本研究旨在探讨抗HPV 16E6核酶（ribozyme)对宫颈癌CaSKi细胞放疗敏感性的影响。方法：以脂质体法将抗HPV 16E6－ribozyme,空载体质粒分别导入CaSKi细胞,命名为CaSKi-R,CaSKi-P细胞。点杂交检测核酶在细胞中的表达,Northern杂交检测3种细胞中E6基因的表达。用克隆形成试验检测3种细胞对放疗的敏感性,Annexine/PI双标法检测细胞凋亡率,流式细胞术检测bcl-2,p53,bax蛋白表达。结果：点杂交证实核酶能在CaSKi－R细胞中,Northern杂交证实CaSKi－R中E6表达较CaSKi－P,CaSKi中明显降低。CaSKi－R细胞生长速度较CaSKi－P,CaSKi明显减慢（P＜0．01）,CaSKi－R细胞对X射线的敏感性较CaSKi－P,CaSKi明显增加,克隆形成率明显下降（P＜0．05）,CaSKi－R细胞照射前后的凋亡率较X射线照射后CaSKi－P,CaSKi明显增加（P＜0．01）;CaSKi－R细胞p53,bax蛋白表达较CaSKi－P,CaSKi明显升高,bcl-2明显减少（P＜0．01）。结论：转染抗HPV16E6－ribozyme的CaSKi-R细胞出现一定程度的生长抑制,且对放射治疗的敏感性增加。     

抗HPV16E6核酶对宫颈癌细胞端粒酶活性的影响及其机制

目的：研究抗HPV16E6核酶（Ribozyme）对宫颈癌CaSKi细胞端粒酶活性的抑制及其机制。方法：以脂质体法将抗HPV16E6-Ribozyme、空载体质粒分别导入CaSKi细胞,命名为CaSKi-R、CaSKi-P细胞。点杂交检测核酶在细胞中的表达,Western blotting法检测3种细胞HPV16E6蛋白的表达,用TRAP-ELISA法检测端粒酶活性,用RT-PCR法检测P53、c-myc、hTERT和hRT的表达。结果：点杂交证实核酶能在CaSKi-R细胞中稳定表达,Western blotting证实CaSKi-R中表达E6蛋白较CaSKi-P、CaSKi明显降低。CaSKi、CaSKi-P、CaSKi-R3种细胞的端粒酶活性值分别为（0.89±0.14）、（0.90±0.11）、（0.36±0.06）,转染了抗HPV16E6核酶的CaSKi-R细胞端粒酶活性的抑制率为59.55%,与CaSKi、CaSKi-P细胞比较明显下降（P〈0.01）。与CaSKi和CaSKi-P细胞相比,CaSKi-R细胞表达c-myc、hTERT mRNA明显减少,P53、hRT mRNA表达无明显变化。结论：抗HPV16E6核酶明显抑制CaSKi-R细胞端粒酶活性,其机制可能与HPV16的E6蛋白及c-myc、hTERT mRNA减少有关。     

靶向切割HPVl6E6 mRNA的核酶诱导宫颈癌细胞CaSKi凋亡的研究

背景与目的：人乳头瘤病毒与宫颈癌的发生相关,核酶是一种能切割靶RNA的特殊RNA。本研究旨在探讨转染了靶向切割HPVl6E6 mRNA核酶的宫颈癌细胞株的表型,以及核酶对宫颈癌细胞增殖与调亡的作用。方法：计算机设计特异性切割HPVl6E6 mRNA的核酶,构建抗HPVl6E6核酶(HRz)的真核表达质粒。以脂质体法将抗HPVl6E6核酶、空载体质粒分别导入CaSKi细胞,命名为CaSKi—R和CaSKi—P细胞。Northern杂交检测3种细胞中E6基因的表达。流式细胞仪检测3种细胞的凋亡率,将3种细胞在相差显微镜和荧光显微镜下观察,采用荧光(Hoechst33342)染色和TUNEL(TDT—mediated dUTP nick end labeling)两种方法测定细胞凋亡。流式细胞术测定HPVl6E6、c—myc、bcl—2、p53、Fas等蛋白的表达。结果：RNA点杂交证实核酶mRNA能稳定表达于CaSKi—R细胞中。Northern杂交证实CaSKi—R中E6mRNA表达水平较CaSKi细胞明显降低,而CaSKi—P和CaSKi细胞的E6mRNA表达水平无差异。CaSKi—R细胞凋亡率明显高于CaSKi和CaSKi—P细胞,细胞周期阻滞于C期,S期细胞百分率下降。抗HPVl6E6核酶能明显减少CaSKi—R细胞中HPVl6E6、c—myc、bcl—2蛋白的表达,增高P53蛋白的表达;这种现象并不发生于CaSKi—P细胞中。Fas蛋白的表达在3种细胞中都相近。结论：抗HPVl6E6核酶的导入能诱导宫颈癌细胞凋亡,其原因可能在于病毒癌基因E6表达的降低,以及由此而引起的细胞内一系列基因表达的改变,包括c—myc、bcl—2、p53等基因。     

宫颈癌中癌基因、细胞增殖和细胞凋亡的定位观察

宫颈癌的发展缓慢且多由癌前病变发展而来。本研究采用免疫组化技术及ＤＮＡ缺口末端标记(ＴＵＮＥＬ)技术对正常宫颈、宫颈上皮内瘤样病变(ＣＩＮ)和宫颈鳞癌患者的手术石蜡标本进行癌基因、细胞增殖和细胞凋亡状况的检测 ,同时对该组患者的临床及病理资料进行分析。1　材料与方法1.1　材料取自上海医科大学妇产科医院 1987年 1月至1994年 12月手术石蜡标本 4 7例 ,其中ＣＩＮⅠ～Ⅱ级 8例 ,ＣＩＮⅢ级 16例 ,宫颈鳞癌 18例 ,年龄 2 9～ 73岁 ,平均 56岁。同期同年龄段正常宫颈组织 5例作对照。1.2　方法检测ｐ53、ｃ ｍｙｃ表达和ＰＣＮＡ…     

热疗对人宫颈癌Hela细胞凋亡的影响

目的:观察不同加温温度对人宫颈癌Hela细胞凋亡的影响.方法:人宫颈癌Hela细胞株常规方法培养,采用水浴加热法(温度为41℃、42.5℃、43.5℃、)对细胞进行加温处理,处理后继续培养24h.用流式细胞仪检测细胞凋亡,单细胞凝胶电泳(single cell gel electrophoresis,SCGE)法检测DNA受损状态.结果:随着温度的增加细胞凋亡率增加,42.5℃、43.5℃加温1h后细胞凋亡率最高分别为30.7%和34.6,坏死细胞分别为13.2%和29.6%.单细胞凝胶电泳发现42.5℃加温1h后40.0%的细胞有DNA损伤,43.5℃加温1h后80.0%以上的细胞DNA损伤,而41℃加温处理1h后仅有20.0%细胞DNA受损.结论:单独加温处理1h可诱导细胞凋亡,并导致细胞DNA损伤.     

Apoptosis Induced by Ginsenoside Rg3 in a Human Bladder Carcinoma Cell Line

OBJECTIVE This study was conducted to explore the effect of Rg3 on inhibition of proliferation and induction of apoptosis in bladder cancer cells. METHODS The EJ bladder cancer cell line was treated with Rg3 at various concentrations. Cell proliferation was measured by the MTT assay. Morphological changes in the cells were observed by fluorescent staining using Hoechst 33258. The cell cycle and apoptotic rate were analyzed by flow cytometry (FCM) and the expression of caspase-3 in cells was detected by immunocytochemistry. DMA ladder analysis was conducted by agarose gel electrophoresis. RESULTS Rg3 inhibited proliferation of EJ cells in a concentration-dependent manner, resulting in an IC50 for Rg3 at 48 h of 125.5μg/ml. When treated with 150μg/ml of Rg3 for 24 h and 48 h, the cells showed apoptotic morphological characteristics including condensed chromatin, nuclear fragmentation, apoptotic bodies and bright fluorescent granules as well as a higher caspase-3 expression. The FCM assay indicated that Rg3 altered the cell cycle and induced apoptosis of the EJ cells, when treated for 24 h and 48 h with 75μg/ml of Rg3 as well as for 48 h with 150μg/ml. The percentages of cells in the S phase and the G2/M transition were increased, whereas the percentages of cells in the G0-G1 transition were decreased. The apoptotic rates were increased from (1.05±0.17)% in the control group cells to (8.41±0.98)%,(18.57±2.20)% and (33.98±1.64)% respectively. Significant changes in the DNA ladders, showed that the effects of Rg3 were displayed in a dose and time dependent manner. CONCLUSION The results suggest that Ginsenoside Rg3 exerts an inhibitory effect on proliferation of EJ cells by inducing apoptosis.     

端粒酶特异性核酶抑制宫颈癌细胞的作用

目的 :研究端粒酶RNA特异性核酶对宫颈癌细胞活性的影响。方法 :通过脂质体将核酶转染至宫颈癌Hela细胞 ,以G4 18筛选细胞 ,经斑点杂交提示转染成功后 ,检测细胞活力及细胞周期变化 ,测定细胞端粒酶活性 ,观察细胞的增殖情况。结果 :试验组细胞的活力较对照组明显减弱。转染后细胞受阻于G1期的细胞明显增多 ,S期细胞比例明显降低 ,P <0 0 1。转核酶细胞端粒酶活性较对照组明显减低 ,转染空载体的细胞端粒酶活性与未转染细胞比较则无明显变化。结论 :端粒酶RNA特异性核酶可抑制宫颈癌细胞端粒酶活性 ,抑制细胞增殖 ,为核酶用于恶性肿瘤基因治疗提供了试验依据     

Newly Established Uterine Cervical Carcinoma Cell Line with Co-amplification of Human Papillomavirus DNA and c-myc Gene

A new human tumor cell line, NCC-c-CX-1 (CX-1), was established from a uterine cervical cancer xenografted in nude mice. This cell line harbored approximately 50 to 100 copies of human papillomavirus (HPV) type 18 DNA per haploid genome, and contained about 16-fold-amplified c-myc gene with rearrangement. These genomic alterations found in CX-1 cells were also present in both primary tumor and xenografted tumor. Histopathologically, original and xenografted tumors were poorly differentiated cancer and were characterized by neuroendocrine features such as positive neuron-specific enolase and chromogranin A by immunohistochemistry and abundant neurosecretory-type granules in the cytoplasm by electron microscopy. However, the established cell line had lost the neuroendocrine features. This cervical cancer cell line may be a useful model for studying cervical carcinogenesis, especially the interaction between HPV and c- myc oncogene.     

冬凌草甲素诱导结肠癌细胞凋亡的实验研究

目的　观察冬凌草甲素体外诱导人结肠癌HCT8细胞凋亡的作用。方法　利用光镜及流式细胞仪观察不同浓度冬凌草甲素诱导结肠癌HCT8细胞凋亡的作用。结果　冬凌草甲素可诱导HCT8细胞凋亡 ,且凋亡率随着浓度的增加而增加。结论　冬凌草甲素对HCT8细胞株具有体外抗肿瘤作用 ,其作用机制与诱导凋亡有关     

Cervical cancer prevention

Cervical cancer is the second most common female tumor worldwide, and its incidence is disproportionately high (>80%) in the developing world. In the United States, in which Papanicolaou (Pap) tests have reduced the annual incidence to approximately 11,000 cervical cancers, >60% of cases are reported to occur in medically underserved populations as part of a complex of diseases linked to poverty, race/ethnicity, and/or health disparities. Because carcinogenic human papillomavirus (HPV) infections cause virtually all cervical cancer, 2 new approaches for cervical cancer prevention have emerged: 1) HPV vaccination to prevent infections in younger women (aged ≤18 years) and 2) carcinogenic HPV detection in older women (aged ≥30 years). Together, HPV vaccination and testing, if used in an age‐appropriate manner, have the potential to transform cervical cancer prevention, particularly among underserved populations. Nevertheless, significant barriers of access, acceptability, and adoption to any cervical cancer prevention strategy remain. Without understanding and addressing these obstacles, these promising new tools for cervical cancer prevention may be futile. In the current study, the delivery of cervical cancer prevention strategies to these US populations that experience a high cervical cancer burden (African‐American women in South Carolina, Alabama, and Mississippi; Haitian immigrant women in Miami; Hispanic women in the US‐Mexico Border; Sioux/Native American women in the Northern Plains; white women in the Appalachia; and Vietnamese‐American women in Pennsylvania and New Jersey) is reviewed. The goal was to inform future research and outreach efforts to reduce the burden of cervical cancer in underserved populations. Cancer 2010. © 2010 American Cancer Society.     

Knowledge of Cervical Cancer and Human Papillomavirus among Japanese Women

Background: The combination of human papillomavirus (HPV) vaccination and cervical cancer tests are globally recommended. Although knowledge regarding cervical cancer and HPV and experience of HPV vaccination are reportedly closely associated, the associations between knowledge and frequency of cervical cancer testing are unclear. Methods: We conducted a questionnaire survey regarding the knowledge of cervical cancer and HPV and experience of HPV vaccination and frequency of cervical cancer testing including cervical cytology and HPV testing. Results: Among 99 women who visited Tsuruha Festa, most of the 77 non-medical workers who received information on cervical cancer and HPV through the Internet were not more likely to have knowledge about cervical cancer and HPV than were 12 medical workers who had gotten information in vocational school or university curriculum. The rates of HPV vaccination, cervical cytology, and HPV testing were 4.0%, 14.1%, and 4.0%, respectively, among participants and did not differ significantly according to participant job and age. Knowledge about cervical cancer and HPV was associated with experience of HPV vaccination and frequency of cervical cytology and was not associated with frequency of HPV testing. Conclusions: We observed insufficient knowledge about cervical cancer and HPV among non-medical workers as well as low HPV vaccination, cervical cytology, and HPV testing rates, and knowledge about cervical cancer and HPV to which frequency of cervical cancer testing were partially related. Therefore, the government should take measures to enhance public awareness about cervical cancer and HPV through social media such as the Internet.