【作者】 张萍；

【导师】 王波；

【作者基本信息】 山东大学 ， 妇产科学， 2009， 博士

【摘要】 目的研究血管内皮生长因子C（vascular endothelial growth factor C,VEGF-C）mRNA在宫颈癌、原位癌及正常宫颈组织中的表达情况,分析其在宫颈癌生长与转移中的可能作用。方法应用实时荧光定量PCR法检测并分析48例宫颈癌组织、48例原位癌组织及36例正常宫颈组织标本中VEGF-CmRNA的表达情况,分析其与肿瘤的组织病理类型、病理分化程度、淋巴结转移、肿瘤直径、临床分期之间的关系。结果VEGF-CmRNA在宫颈癌组织、宫颈原位癌组织及正常宫颈组织中的表达量有显著性差异（F=19.21,P＜0.05）,VEGF-C mRNA在宫颈癌组织中的表达显著高于宫颈原位癌组织中的表达（q=9.13,P＜0.05）;VEGF-C mRNA在宫颈原位癌组织中的表达量明显高于正常宫颈组织中的表达（q=4.25,P＜0.05）。宫颈癌组织中VEGF-C mRNA的表达与肿瘤的组织病理类型相关（F=12.01,P＜0.05;q腺癌/鳞癌=3.97,P＜0.05;q腺癌/腺鳞癌=6.35,P＜0.05;q鳞癌/腺鳞癌=3.50,P＜0.05）;宫颈癌组织中VEGF-C mRNA的表达与病理分化程度相关（F=9.76,P＜0.05;qG1/G2=4.01,P＜0.05;qG1/G3=5.33,P＜0.05;qG2/G3=4.24,P＜0.05）;宫颈癌组织中VEGF-C mRNA的表达与淋巴结转移（t=44.15,P＜0.05）相关;宫颈癌组织中VEGF-C mRNA的表达与肿瘤直径（t=39.84,P＜0.05）相关;但宫颈癌组织中VEGF-C mRNA的表达与与宫颈癌的临床分期无明显的相关性（F=2.12,P＞0.05）。结论（1）宫颈癌组织中VEGF-C mRNA表达水平明显高于宫颈原位癌组织及正常宫颈组织,且与临床病理之间有良好的相关性,提示其在宫颈癌生长、转移和预后过程中起着十分重要的作用。（2）VEGF-C可能成为治疗宫颈恶性肿瘤的一个潜在的分子靶点。目的探讨血管内皮生长因子C在宫颈癌细胞生长、增殖中的可能作用。方法采用基于TaqMan探针技术的实时荧光定量PCR法检测体外培养的宫颈癌细胞系Hela细胞、SiHa细胞中VEGF-C mRNA的表达。结果人宫颈癌Hela细胞株中VEGF-CmRNA的表达明显高于人宫颈癌SiHa细胞株中的表达,差别有统计学意义（t=7.1,P＜0.05）。结论VEGF-C在不同增殖能力的宫颈癌细胞株中含量不同,该基因可能与宫颈癌细胞侵袭能力有关,抑制宫颈癌中VEGF-C的表达有望可以抑制宫颈癌的侵袭转移。目的研究针对VEGF-C的小分子干扰RNA（siRNA）介导的RNA干扰（RNAi）技术对人宫颈癌Hela细胞及SiHa细胞中VEGF-C基因及蛋白的靶向抑制作用,同时观察对细胞生长、增殖的影响及形态的变化,探讨针对VEGF-C的siRNA介导RNA干扰技术在宫颈癌基因治疗中的应用前景。方法根据siRNA序列设计原则,设计并合成三条VEGF-C序列特异性小分子干扰RNA（siRNA）及一条阴性对照siRNA,应用德国QIAGEN公司RNAiFect^TM Transfection试剂盒转染人宫颈癌Hela细胞及SiHa细胞。筛选出最有效的siRNA干扰序列后,分别于转染后12h、24h、48h、72h,应用实时荧光定量PCR方法检测Hela细胞及SiHa细胞中VEGF-C mRNA的表达,Western Blot方法检测VEGF-C蛋白的表达,MTT及细胞计数法检测siRNA转染对培养细胞生长增殖的影响。结果1.细胞转染人宫颈癌Hela细胞及Siha细胞转染针对VEGF-C的siRNA 6h后于高倍镜下均可见细胞形态发生改变,轮廓不规则,空泡化形成,部分细胞脱落,有细胞碎片形成,细胞数目较对照组明显减少,通过荧光标记siRNA转染后发现转染效率可达85%以上,摄入的siRNA主要位于细胞浆,围绕胞膜排列;空白对照组及阴性对照组细胞形态正常,细胞透明,细胞贴壁状况良好,呈棱形或多角形,数量较转染实验组明显增多。阴性对照组加入转染反应液后细胞形态也发生改变,但换液后,细胞形态很快恢复正常。2.荧光定量PCR检测RNAi后Hela细胞中VEGF-C mRNA表达统计学分析表明,干扰12-72h时间内,实验组与阴性对照组VEGFmRNA的表达量相比明显降低,差异有显著性意义（q值依次为6.44、8.75、7.34、19.23,P＜0.05）;而阴性对照组及空白组VEGF-C mRNA的表达的差异在不同时相均无显著性（P＞0.05）,干扰效果变化明显。3.荧光定量PCR检测RNAi后Siha细胞中VEGF-C mRNA表达转染12、24、48及72h后,实验组VEGF-CmRNA的表达水平均明显下降,与空白对照组相比,差异有统计学意义（q值分别为16.26、21.44、34.67、15.32,P＜0.05）;与阴性对照组比较差异有统计学意义（q值分别为15.35、20.62、30.24、17.04,P＜0.05）;而阴性及空白对照组VEGF-CmRNA的相对表达量在不同时间点比较差异无统计学意义（q值分别为2.02、1.98、1.43、1.08,P＞0.05）。4.Western Blot检测RNAi后Hela细胞中VEGF-C蛋白的表达DAB显色后发现在46.9KD处有一明显条带,与VEGF-C蛋白大小一致。应用图像分析系统对western blot结果进行半定量分析,空白对照为1,不同干扰时间的数值结果与其进行灰度比较。统计学分析发现与对照组相比,RNAi干扰后目的siRNA对VEGF-C蛋白表达均有不同程度的影响,且干扰时间越长,效果越明显,干扰后同一时相各组VEGF-C蛋白的表达有显著性差异（P＜0.05）,干扰后同一时相阴性对照组与空白组之间的差别无统计学意义（t分别为1.01、0.83、0.71、0.42,P均＞0.05）。5.Western Blot检测RNAi后Siha细胞中VEGF-C蛋白的表达结果显示干扰后12-72h内,实验组VEGF蛋白含量明显低于各对照组,差异具有显著性的意义（P＜0.05）,而阴性对照组和空白组相比则差异无统计学意义（t值分别为0.09,0.49,0.80,1.02,P＞0.05）。6.MTT法检测针对VEGF-C的RNAi后Hela细胞的增殖情况MTT法检测表明针对VEGF-C的siRNA对Hela细胞生长有明显的抑制作用,转染后同一时段与空白对照组及阴性对照组细胞有显著性差异（F值分别10.58、16.17、23.39、41.94,P均＜0.05）,转染后阴性对照siRNA12h-72h后对Hela细胞生长增殖无明显抑制作用,与同一时段空白对照组之间比较差异无显著性（t值分别为0.12、0.24、0.06、0.09;P均＞0.05）。7.MTT法检测siRNA对SiHa细胞增殖的影响目的siRNA对细胞生长有明显的抑制作用,不同时段与各对照组细胞相比差异有统计学意义（F值分别为12.94、32.55、29.45、23.30,P＜0.05）;阴性对照siRNA对细胞生长无明显抑制作用,且不同时段与空白对照组相比差异无统计学意义（q值分别为0.75、1.09、0.90、0.60,P＞0.05）。8.细胞计数法检测针对VEGF-C的RNAi后Hela细胞的数量细胞计数法检测表明目的siRNA对细胞数量有明显影响,不同时间段细胞数与空白对照组相比明显减少（F值分别为4.01、5.32、7.89、13.47,P＜0.05）,阴性对照siRNA组对细胞数量无明显影响,不同时段细胞数与空白对照组之间相比差异无显著性（t值分别为0.71、0.49、0.14、0.93,P＞0.05）。9.细胞计数法检测针对VEGF-C的RNAi后Siha细胞的数量细胞计数法检测表明转染后12h-72h各组Siha细胞数均不相同（F值分别为4.23、6.68、7.12、12.1,P均＜0.05）,实验组细胞数明显低于其余各组,差别有统计学意义;空白组、阴性对照组之间差别无统计学意义（t=0.48、0.24、0.38、1.23,P均＞0.05）。结论（1）针对VEGF-C的siRNA可以有效抑制宫颈癌细胞VEGF-CmRNA及其蛋白表达量,抑制细胞生长,表明VEGF-C基因在宫颈癌的发生、发展中具有重要作用。（2）针对VEGF-C的siRNA介导的RNA干扰技术对高表达VEGF-C的宫颈腺癌Hela细胞及低表达VEGF-C的宫颈鳞癌Siha细胞均可引起显著的靶向基因抑制作用。（3）针对VEGF-C的RNA干扰技术为宫颈癌基因治疗提供了新策略,为下一步利用RNA干扰技术抑制肿瘤血管形成以及局部侵袭和远处转移提供研究基础。更多还原

【Abstract】 PartⅠEXPRESSIONS OF VASCULAR ENDOTHELIAL GROWTH FACTOR-C IN CERVICAL CANCER AND ITS CLINICOPATHOLOGICAL SIGNIFICANCEObjective To investigate the expression of vascular endothelial growth factor C(VEGF-C) mRNA in cervical cancer tissues /para-tumour and normal cervix tissues.The relationships between related mRNAs expression and the tumor metastasis,prognosis were analyzed.Methods Cervical cancer tissues(n=48)、para-tumour tissue(n=48) and their normal controls(n=36)were collected.Using TaqMan real-time fluorescent quantitative PCR method to detect the expressions of VEGF-C mRNA,To analyze the conjunction of the factors’ expressions with the clinical features of the tumor.Results Through real-time fluorescent quantitative PCR method,we funnd that there was a significant difference levels of VEGF-CmRNA expression between the tissues of tumor,para-tumour tissue and that of the normal controls(P＜0.05). The levels of VEGF-C mRNA expression in invasive carcinoma of cervix was higher than that in all matched.And there was no significant relationship between the levels of VEGF-C mRNA expression with the clinical stagein invasive carcinoma of cervix (P＞0.05).But the levels of VEGF-C mRNA expression was significant associated with the pathological types,the grade of tumor pathology,lymph node metastasis, tumor size and the invasion of deep muscular layer(P＜0.05).Conclusion The method of detection for the VEGF-C mRNA expression in cervical cancer tissues by real-time fluorescent quantitative RT-PCR has a high sensitivity and specificity.It is reliable and easily performed.The results suggest that the VEGF-C may play an important role in genesis and development of invasive carcinoma of cervix.It would be a useful biological marker to foresee the lymph node metastasis and monitor the progression of invasive carcinoma of cervix.VEGF-C might be used as a potential molecular target for the treatment of malignant tumors. PartⅡTO DETECT THE EXPRESSION OF VASCULAR ENDOTHELIAL GROWTH FACTOR C mRNA IN CERVICAL CANCER CELL LINES HELA CELLS AND SiHa CELLS BY REAL-TIME QUANTITATIVEPCRObjective To investigate the relationship of vascular endothelial growth factor C and the growth and proliferation of cervical cancer.Methods Cervical cancer cell lines,Hela cell lines and SiHa cell lines,were cultured and detected the contents of VEGF-C mRNA by TaqMan real-time fluorescent quantitative RT-PCR expression.Results The levels of VEGF-C mRNA expression in Hela cell lines was higher significantly than that in SiHa cell line(t=7.1,P＜0.05).Conclusions VEGF-C gene may be related to ability of invasion of cervical cancer,inhibiting the expression of VEGF-C is expected to has the function of inhibiting the invasion and metastasis of cervical cancer. PartⅢTHE EFFECTS OF SiRNA TARGET VEGF-C ON BOTH THE EXPRESSION OF VEGF-C AND THE CELLS PROLIFERATION OF CERVICAL CANCERObjective To study the effects of small interference RNA on VEGF-C mRNA expression in vitro on cultured Hela cell lines and Siha cell lines,the observation of proliferation of the cervical cancer cells,and the exploration of the application of RNA interference mediated by VEGFC-siRNA on cervical cancer gene therapy in the future.Methods On the basis of the principle of target sequence of siRNA,three interference sequences of VEGF-C were designed.The best siRNA interference sequences was screened and then transfected into VEGF-C overexpressed cervical cancer cell line,Hela cells in vitro,and VEGF-C low-expressed cervical cancer cell line,Siha cells in vitro,by siRNAs-liposome complex.VEGF-C mRNA level was detected by real time RT-PCR after transfection by siRNA and began to observe and measure the results of RNA interference in 12 hours.The VEGF-C protein was analyzed by Western blot between transfection and controls.MTT method and cell count methods were used to observe the effects of RNAi on the bioactive behalves of cells.Results After transfected,the cells in the experimental group,deformate, shrink,and began falling out.The growth of Hela cells and Siha cells were both obviously inhibited,Whereas the cells in control groups had not obviously changed. Sequence specific siRNAs of VEGF-C effectively downregulated the mRNA level of VEGF-C.Western blot revealed that the protein levels were down regulated obviously post transfection compared with the control in 12h/24h/48h and 72h(P＜0.05).They were significantly different compared with the control groups.During 12h-72h after transfected,the inhibition ratio of proliferation in both Hela cells and Siha cells were obviously higher than the controls’(P＜0.05).There was a significant difference in the levels of cell population of experimental group and the controls during 12h-72h(P＜0.05).Conclusions The small interference RNA target VEGF-C can effectively downregulate VEGF-C on both mRNA and protein,inhibit the growth of Hela cells and Siha cells.It hints that VEGF-C may plays an important role in the development of cervical cancer,which provides an experimental basis for treating human tumors with anti-angiogenesis method using RNAi..更多还原