抗HPV16E6核酶对宫颈癌CaSKi细胞增殖和侵袭力的影响

目的 研究特异性抗HPV16E6核酶对宫颈癌CaSKi细胞增殖和侵袭能力的影响。方法 以脂质体法将抗HPV16E6核酶、空载体质粒分别导入CaSKi细胞,分别为CaSKi-R和CaSKi-P。观察细胞的生长状态,测定CaSKi,CaSKi-R和CaSKi-P3种细胞的生长曲线、软琼脂克隆形成率、细胞侵袭力实验、裸鼠体内致瘤性,以及RT-PCR检测细胞中COX-2、血管内皮生长因子(VEGF)的表达,同时,免疫组化检测3种细胞COX-2、VEGF抗原表达,分析特异性抗HPV16E6核酶对宫颈癌CaSKi细胞增殖和侵袭力的影响。结果 CaSKi、CaSKi-P细胞生长速率、软琼脂克隆形成率、致瘤性和侵袭力相近,而CaSKi-R细胞的细胞生长速率、软琼脂克隆形成率、致瘤性和侵袭力与以上两种细胞相比却明显降低。RT-PCR检测CaSKi-R细胞的COX-2、VEGF表达水平也低于CaSKi、CaSKi-P细胞的表达。细胞免疫组化测定CaSKi、CaSKi-P和CaSKi-R细胞都有COX-2、VEGF抗原表达,而CaSKi-R细胞的抗原染色程度较弱。结论 特异性抗HPV16E6核酶的导入降低了宫颈癌CaSKi细胞的增殖和侵袭表型,可能与宫颈癌CaSKi细胞内COX-2、VEGF的表达水平降低有关。     

抗HPV16E6核酶对宫颈癌CaSKi细胞增殖和侵袭力的影响

目的研究特异性抗HPV16E6核酶对宫颈癌CaSKi细胞增殖和侵袭能力的影响。方法以脂质体法将抗HPV16E6核酶、空载体质粒分别导入CaSKi细胞，分别为CaSKi-R和CaSKi—P。观察细胞的生长状态，测定CaSKi．CaSKi—R和CaSKi—P3种细胞的生长曲线、软琼脂克隆形成率、细胞侵袭力实验、裸鼠体内致瘤性，以及RT—PCR检测细胞中COX-2、血管内皮生长因子(VEGF)的表达，同时，免疫组化检测3种细胞COX-2、VEGF抗原表达．分析特异性抗HPV16E6核酶对宫颈癌CaSKi细胞增殖和侵袭力的影响：结果CaSKi、CaSKi—P细胞生长速率、软琼脂克隆形成率、致瘤性和侵袭力相近，而CaSKi-R细胞的细胞生长速率、软琼脂克隆形成率、致瘤性和侵袭力与以上两种细胞相比却明显降低。RT—PCR检测CaSKi—R细胞的COX-2、VEGF表达水平也低于CaSKi、CaSKi—P细胞的表达。细胞免疫组化测定CaSKi、CaSKi—P和CaSKi—R细胞都有COX-2、VEGF抗原表达，而CaSKi—R细胞的抗原染色程度较弱。结论特异性抗HPV16E6核酶的导入降低了宫颈癌CaSKi细胞的增殖和侵袭表型，可能与宫颈癌CaSKi细胞内COX-2、VEGF的表达水平降低有关。     

特异性核酶对宫颈癌细胞系CaSKi增殖与凋亡的影响

目的研究特异性抗HPV16E6核酶对宫颈癌细胞增殖与凋亡的影响。方法设计特异性切割HPV16E6基因的核酶,构建抗HPV16E6核酶的真核表达质粒。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、CaSKi-P细胞。点杂交检测核酶在细胞中的表达,Northern blotting检测CaSKi、CaSKi-R、CaSKi-P 3种细胞中E6基因的表达。测定3种细胞的生长曲线和软琼脂克隆形成率,并以皮下接种法检测细胞在裸鼠体内的成瘤能力。流式细胞仪检测3种细胞的凋亡率,并测定c-myc、bcl-2、p53、fas、PCNA、C-erbB-2等基因的表达。结果点杂交证实该核酶能在CaSKi-R细胞中稳定表达,Northern blotting证实CaSKi-R中表达E6较CaSKi-P、CaSKi明显降低。与CaSKi细胞相比,CaSKi-R细胞生长速度、软琼脂克隆形成率明显降低,在裸鼠体内的致癌能力下降,凋亡率明显增高,出现凋亡峰,S期、G2M期细胞百分率下降;而CaSKi-P细胞无此改变。CaSKi-R细胞表达HPV16E6、C-myc、Bcl-2、PCNAC-erbB-2蛋白明显减少,而p53表达明显增高;CaSKi和CaSKi-R细胞中Fas 蛋白的表达相近。CaSKi-P细胞中各基因的表达与CaSKi细胞无显著差异。结论抗HPV16E6核酶的导入能阻碍宫颈癌细胞增殖,诱导其凋亡,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     

抗人乳头瘤病毒16型E6基因核酶对宫颈癌CaSKi细胞生长和端粒酶活性的影响

目的研究抗HPV16E6核酶(Ribozyme)对宫颈癌CaSKi细胞生长和端粒酶活性的影响.方法以脂质体法将抗HPv16E6-Ribozyne、空载体质粒分别导入CaSKi细胞,命名为CaSKi-R和CaSKi-P细胞.点杂交检测核酶在细胞中的表达,northern杂交检测三种细胞中E6基因的表达;细胞生长曲线检测细胞生长的改变;TRAP-Elisa法检测端粒酶活性.结果点杂交证实核酶能在CaSKi-R细胞中稳定表达,northern杂交证实CaSKi-R中表达E6较CaSKi-P和CaSKi明显降低.CaSKi-R细胞生长速度较CaSKi-P和CaSKi明显减慢(P＜0.01);CaSKi,CaSKi-P和CaSKi-R三种细胞的端粒酶活性分别为0.89±0.14,0.90±0.11,0.36±0.06,转染了抗HPV16E6核酶的CaSKi-R的端粒酶活性的抑制率为59.55%.经统计学处理,CaSKi-R的端粒酶活性较CaSKi和CaSKi-P细胞明显下降(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,端粒酶活性较前明显下降.     

抗HPV16E6核酶对宫颈癌CaSKi细胞化疗的影响

目的研究抗HPV16E6核酶对宫颈癌CaSKi细胞化疗敏感性的影响。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。点杂交检测核酶在细胞中的表达,Northern杂交检测三种细胞中E6基因的表达。用克隆形成试验检测3种细胞对化疗的敏感性,Annexine/PI双标法检测细胞凋亡率。结果点杂交证实核酶能在CaSKi-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。CaSKi-R细胞的生长速率较CaSKi-P、CaSKi明显减慢,泰素作用后相对克隆形成率和凋亡率在3种细胞间亦无差异(P>0.05);顺铂作用后CaSKi-R细胞的相对克隆形成率较CaSKi-P、CaSKi明显下降(P<0.01),而凋亡率明显增加(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,且对顺铂的敏感性增加,而对泰素的敏感性没有发生改变。     

抗HPV16E6核酶对宫颈癌CaSKi细胞放疗影响的研究

目的人乳头瘤病毒（HPV）是官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；放射治疗是目前宫颈癌治疗的标准和有效的方法；该研究旨在探讨抗HPV16E6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。点杂交交检测核酶在细胞中的表达，Northem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细胞中稳定表达，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．（31）；CaSKi—R细胞p53、bax蛋白表达较CaSKi—P、CaSKi明显升高山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，且时放射治疗的敏感性增加。     

抗HPV16E6核酶对宫颈癌CaSKi细胞化疗的影响

目的：研究抗HPV16E6核酶对宫颈癌CaSKi细胞化疗敏感性的影响。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。点杂交检测核酶在我的表达，Northern杂交检测三种细胞中E6基因的表达。用克隆形成试验控制3种细胞对化疗的敏感性，Annexine/PI双标法检测细胞凋亡率。结果：点杂交证实核酶能在CaSKi－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。CaSKi－R细胞的生长速率较CaSKi－P，CaSKi明显减慢，泰索作用后相对克隆形成率和凋亡率在3种细胞间亦无差异（P＞0．05）；顺铂作用后CaSKi－R细胞的相对克隆形成率较CaSKi－P，CaSKi明显下降（P＜0．01），而凋亡率明显增加（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，且对顺铂的敏感性增加，而对泰素的敏感性没有发生改变。     

特异性核酶对宫颈癌细胞系CaSki增殖与凋亡的影响

目的：研究特异性抗HPV 16E6核酶对宫颈癌细胞增殖与凋亡的影响。方法：设计特异性切割HPV 16E6基因的核酶，构建抗HPV16E6核酶的真核表达质粒。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、CaSki－P细胞。点杂交检测核酶在细胞中的表达，Northern blotting检测CaSki、CaSki－R、CaSki－P3种细胞中E6基因的表达。测定3种细胞的生长曲线和软琼脂克隆形成率，并以皮下接种率法检测细胞在裸鼠体内的成瘤能力。流式细胞仪检测3种细胞的凋亡率，并测定c-myc、bcl-2、p53、fas、PCNA、C－erbB-2等基因的表达。结果：点杂交证实该核酶能在CaSki－R细胞中稳定表达，Northern blotting证实CaSki－R中表达E6较CaSki－P、CaSki明显降低，出现凋亡峰，S期、GWMS期细百分率下降；而CaSki－P细胞无此改变。CaSki－R细胞表达HPV16E6、C－myc、Bcl-2、PCNA、C－erbB-2蛋白明显减少，而p53表达明显增高；CaSki和CaSki－R细胞中Fas蛋白的表达相近。CaSki－P细胞中各基因的表达与CaSki细胞无显著差异。结论：抗HPV16E6核酶的导入能阻碍宫颈癌细胞增殖，诱导其凋亡，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。     

Transfection with the ribozyme targeting HPVE6 mRNA results in growth inhibi-tion of E6-expressing cervical carcinoma cells

To acquire a ribozyme against the E6 gene of human papillomaviruses type 16 ( HPV16E6 ) and investigate its effects on the phenotypes and gene expression of cervical cancer cell line. Methods: Anti-HPV16E6 ri-bozyme (HRz) was designed by computer programs and its activity identified by cleavage experiment in vitro before its transfection v/a lipofectin into CaSKi cells with the empty eucaryotic expression plasmid transfection of the cells also performed, the resultant cells designated as CaSKi-R, CaSKi-P respectively. The morphology and the soft agar forming ability were studied in Ca,SKi cells and the transfected cells, and the expression of E6, proliferating cell nuclear antigen (PCNA) and C-erbB-2 genes assayed by flow cytometry. The tumorgenicity of each cell line was evaluated in nude mice receiving inoculations of CaSKi, CaSKi-R and CaSKi-P cells separately, while in one group, both CaSKi and CaS-Ki-R cells were inoculated on different sides of the mice. Results: HRz was able to cleave HPV16E6 mRNA in a site-specific manner and could be expressed stably in transfected CaSKi ceils. Northern blot analysis showed that E6 mRNA was less in CaSKi-R than in CaSKi cells, and no significant difference in the morphology and growth rate was observed between CaSKi and CaSKi-P cells, but the growth rate CaSKi-R was lowered. The colony-forming rate of CaSKi-P insoft agar was similar to that of Ca,SKi cells, while that of CaSKi-R was decreased. Flow cytometry showed that anti-HPV16E6 ribozyme reduced the expression of E6, PCNA and C-erbB-2 genes in CaSKi-R cells, but not in CaSKi-Pcells. The tumorgenicity of CaSKi-R in nude mice was decreased compared with Ca,SKi cells. Conclusion: HRz canpartially reverse the malignant phenotype of Ca,SKi cells, possibly due to decreased E6 gene expression, and the conse-quent decrease of PCNA and C-erbB-2 gene expressions.     

人乳头瘤病毒16型E5对人宫颈癌SiHa细胞恶性潜能的影响及其机制探讨

目的 通过构建和筛选持续表达HPV16E5的细胞SiHa/16E5,探讨HPV16E5对宫颈癌SiHa细胞的作用及机理.方法 利用pEGFP-C1构建HPV16型E5的正义全长真核表达载体并稳定转染SiHa细胞;利用RT-PCR和Western印迹技术检测转染前后E5和P21基因的mRNA和GFP+E5和P21蛋白的变化;利用MTT法检测SiHa细胞稳定转染后的增殖活性;利用软琼脂克隆形成和裸鼠成瘤实验分别在体内外验证了SiHa/16E5细胞的致瘤情况.结果 稳定转染携带.HPV16全长E5基因的质粒后,SiHa细胞中E5基因的mRNA和相应蛋白表达明显升高,而P21的表达则明显下调;MTT结果 显示稳定转染后细胞的增殖活性与转染空载体细胞和未转染细胞比较明显增高(P<0.05);软琼脂克隆形成结果 示:SiHa/16ES细胞组的可隆形成数为33.4±1.6,与空质粒对照组细胞(15.1±3.1)及未转染组细胞(16.3±2.5)比较差异有统计学意义(P<0.05);裸鼠成瘤实验结果 显示,4个实验组在共20 d的观察中,SiHa/16E5组裸鼠成瘤明显快于其他3个对照组(P<0.05).结论 HPV16 E5可降低宫颈癌SiHa细胞中P21的表达,加强宫颈癌SiHa细胞增殖和成瘤效应.     

Effects of anti-HPV16 E6-ribozyme on the proliferation and apoptosis of human cervical cancer cell line CaSKi.

OBJECTIVE: To study the characterization of anti-HPV16E6-ribozyme transfected into cultured human cervical cancer cell line, and to investigate the effect of ribozyme on the proliferation and apoptosis of the cells. METHODS: By way of lipofectin transfection, anti-HPV16E6-ribozyme, a ribozyme designed to specifically cleave the HPV16E6 gene, and empty vector expression plasmids were respectively transfected into CaSKi cells which were subsequently designated as CaSKi-R and CaSKi-P accordingly. The expression of ribozyme in the transfected cells was observed by RNA dot blot analysis, and E6 mRNA expression was detected by Northern blotting in the 3 kinds of cells whose growth curves and clone-forming ability on soft agar were studied, with their tumorgenicity observed by percutaneous inoculation of the cells in nude mice. Flow cytometry was employed to assess the apoptosis rates and the expression of the genes including c-myc, bcl-2, p53, and fas etc. RESULTS: Stable expression of anti-HPV16E6-ribozyme was observed in CaSKi-R cells that had less E6 mRNA expression than CaSKi had as shown by Northern blotting. When compared with those of CaSKi cells, the growth rate and clone-forming ability on soft agar of CaSKi-R were reduced, while those of CaSKi-P evinced no significant changes. The tumorgenicity of CaSKi-R in nude mice was also comparatively decreased, with markedly elevated apoptosis rate. Anti-HPV16E6-ribozyme reduced the expressions of E6, c-myc, bcl-2, PCNA and C-erbB2 genes but increased p53 expression in CaSKi-R cells, which, however, did not happen in CaSKi-P cells. The expression of Fas underwent no significant changes in response to the transfection. CONCLUSION: Anti-HPVE6-ribozyme inhibits proliferation and induces apoptosis in CaSKi cells, possibly due to the decrease of E6 gene expression that triggers a series of changes in the gene expressions of the cells.     

Effects of anti- HPV16E6- ribozyme on phenotype and gene expression of a cervical cancer cell line

Objective To investigate the effects of anti- HPV16E6- ribozyme (HRz) on phenotype and gene expression of a cervical cancer cell line. Methods HRz was designed by computer programs.HRz’s activity was identified by cleavage experiments in vitro.HRz and empty eukaryotic plasmids were transfected into CaSKi cells with lipofectin, then renamed CaSKi- R and CaSKi- P, respectively. The expression of ribozyme in transfected cells was observed by RNA dot blot.The amounts of E6 mRNA in three kinds of cells lines were detected by Northern blot.Cell growth curves and soft agar forming ability were studied.The ability of each cell line to form tumors was assessed in nude mice.Apoptosis rates and expression of c- myc, bcl- 2, p53 and Fas were detected by flow cytometry (FCM).Antigens of tumor cells, HLA- 1, HLA- 2, B7- 1 and B7- 2 were also detected.NK, LAK, and CD3AK cells were induced.Their cytotoxicities were detected in CaSKi- R, CaSKi- P, and CaSKi cells. Results In vitro cleavage reaction demonstrated that HRz could cleave HPV16E6 mRNA in a site- specific manner.HRz could be expressed stably in transfected CaSKi cells.Northern blot analysis showed that E6 mRNA levels were lower in CaSKi- R than in CaSKi.The growth rate of CaSKi- R was slower than those of CaSKi and CaSKi- P.The soft agar- forming rate of CaSKi- R was lower compared with those of CaSKi and CaSKi- P cells.The ability of CaSKi- R to form tumors in nude mice was also poor.The apoptosis rate of CaSKi- R cells was much higher than those of CaSKi and CaSKi- P.HRz could reduce the expression of E6, c- myc and bcl- 2 proteins, and increase the expression of p53 as well.HRz could increase the expression of HLA- 2, B7- 1 and B7- 2 antigens.The cytotoxicity of NK, LAK and CD3AK cells was much higher in CaSKi- R than in CaSKi- P and CaSKi cells.Conclusion HRz not only reverses the malignant phenotype of CaSKi cells partially, but also induces apoptosis in the cells, and increases sensitivity of CaSKi cells to immune cells.