【作者】 崔艳国；

【导师】 邢福祺；

【作者基本信息】 南方医科大学 ， 妇产科学， 2008， 博士

【摘要】 妊娠滋养细胞疾病包括完全、部分和侵袭性葡萄胎,胎盘滋养细胞肿瘤以及绒癌。因为人类滋养细胞是有异于体细胞的特殊细胞,具有独特的分化特点和生物学特性,与人类其他的肿瘤细胞相似。因此,研究妊娠滋养细胞疾病的发生、发展机制已成为当前妊娠滋养细胞疾病基础研究的一个重要领域。由于正常绒毛组织体外分离培养的限制,使得经典基因转染研究方法难以实施。所以,我们采用RNA干扰(RNAi)技术从反向遗传学的角度来阐明F10基因的功能。RNA干扰(RNAi,RNA interference)是一种首先在线虫中发现的转录后基因沉默现象(PTGS,post—transcriptional gene silencing),由双链RNA(dsRNA,double-strand RNA)启动,并导致具有同源序列mRNA的降解。2001年,Tuschl的实验室首先报道化学合成的21bp小干扰RNA(siRNA,smallinterfering RNA)在哺乳动物细胞能序列特异地抑制基因表达,又不至于激活宿主细胞的抗病毒反应,使得RNAi技术被证实为一项快速有效的、适宜高通量的基因功能分析技术。反向遗传学技术也因此成为目前研究基因功能的有效手段。在反向遗传学研究中,反义寡核苷酸和核酶技术曾被应用,但其应用程度和效果极低。目前,除同源重组介导的基因敲除(knock-out)外,还没有其他通用的反向遗传学技术。基因敲除技术由于操作费时、费用昂贵及成功率低大大地限制了其广泛应用,并且基因敲除不适于人类细胞。与传统的反义核酸技术相比,通过双链RNA起作用的RNAi具有如下优点:双链RNA比反义核酸更为稳定,不容易被降解;相同量的双链RNA抑制基因表达的效果至少比反义核酸显著;高特异性,靶基因mRNA一个碱基的变异即可使siRNA失去RNAi效果;绝大多数基因的表达都能被双链RNA介导的RNAi所抑制。所以,RNA干扰作为一种新的反向遗传学技术引起广泛关注,并在基因功能的研究中得以应用。肿瘤发生相关基因的异常表达是肿瘤发生的一个重要原因,因此抑制肿瘤相关基因表达的研究成为肿瘤发病机制中的重要方面。2005年,我们前期通过抑制性消减杂交技术,获得了正常早孕绒毛膜组织与葡萄胎组织之间的消减文库,并从中分离出一个在葡萄胎中高表达的新基因—F10;采用原位杂交方法检测12例葡萄胎,6例侵袭性葡萄胎,8例绒癌中F10基因表达情况。结果F10基因在全部葡萄胎、侵袭性葡萄胎、绒癌中均呈阳性表达;其在葡萄胎,侵袭性葡萄胎、绒癌中的表达强度递增。研究证明F10可能与滋养细胞肿瘤的发生及其侵袭行为相关,在侵袭或滋养细胞恶变中起一定作用。2006年,课题组采用原位杂交方法检测不同肿瘤组织和正常组织的F10基因mRNA表达。结果发现F10基因在卵巢腺癌、子宫内膜癌、乳腺癌、肝癌、胃癌等腺癌中阳性表达,不同腺癌之间阳性表达差异无显著性。子宫内膜、宫颈上皮等正常组织、肝癌癌旁组织中F10基因呈阴性表达。上述研究证实:葡萄胎差异表达新基因F10不仅与滋养细胞肿瘤密切相关,可能还在某些腺癌发生发展中起重要作用。葡萄胎差异表达新基因F10是本课题组前期研究中发现的新基因,本课题通过以RNA干扰(RNA interference RNAi)为基础的基因沉默技术对F10基因的功能及机理进行初步研究。第一部分瞬时转染dsRNA确定F10基因沉默对KLE细胞凋亡的影响一研究内容与方法探讨小分子双链RNA对KLE细胞中F10基因的沉默效应,并确定F10基因沉默对KLE细胞凋亡的影响。采用体外转录法制备针对F10基因的dsRNA;脂质体转染KLE细胞,Taqman探针法荧光定量PCR检测dsRNA转染KLE细胞不同时间后细胞中F10基因的表达水平;流式细胞术检测F10基因沉默后KLE细胞凋亡的改变。二结果1、体外转录法能制备足够的dsRNA,丙烯酰胺凝胶电泳见dsRNA分子量为21bp左右。经检测ds RNA浓度为740 ng/μl(52.85μmol/L)2、Taqman探针法荧光定量PCR检测显示:与对照组相比,10nM,20nM和50nM的dsRNA转染KLE细胞后24小时均出现F10基因拷贝数的下调,20nM和50nM的dsRNA转染可导致KLE细胞F10表达显著下调,并且基因下调一直维持到转染后72小时。其中20nM dsRNA转染48小时获得最大的F10基因沉默效应,F10基因表达下调达83%。3、流式细胞术对KLE细胞DNA含量的检测发现:20nM dsRNA转染48小时后,KLE细胞凋亡率升高到8.91%,对照组在转染48小时后细胞凋亡率为0.36%,实验组的凋亡率是对照组细胞的24.75倍。三小结1、通过体外转录法制备针对F10基因的dsRNA;脂质体转染KLE细胞,Taqman探针法荧光定量PCR检测dsRNA转染KLE细胞不同时间后细胞中F10基因的表达水平,提示:体外转录制备针对F10基因的dsRNA能特异有效沉默KLE细胞中的F10基因。2、流式细胞术对KLE细胞DNA含量的检测发现F10基因的表达下调可诱导KLE细胞的凋亡。第二部分沉默载体的构建和稳定克隆的建立一研究内容与方法针对F10基因设计能转录生成发卡样siRNA的寡核苷酸片段,构建出pRetroQ-i F10重组体并建立F10基因表达稳定下调的KLE细胞系。依据GenBank中F10基因(Genbank NO.gi:5641046)的序列,用设计分析软件进行筛选序列,确定能制备针对F10基因siRNA的寡核苷酸序列。合成单链寡核苷酸,片段退火成双链DNA,将两条合成的寡核苷酸退火后形成的双链DNA连接到逆转录病毒载体pRetroQ上构建重组质粒pRetroQ-i F10,脂质体转染KLE细胞,嘌呤霉素筛选,获得单细胞克隆.二结果1对构建的pRetroQ/i F10重组质粒测序结果证实shRNA表达模板成功克隆于pRetroQ载体上,插入序列完全正确无碱基突变和缺失。2对筛选获得的单细胞克隆进行F10基因的荧光定量RT-PCR检测发现:2个RetroQ/negative细胞克隆中F10表达水平没有明显差别;5个pRetroQ/RNAi细胞克隆出现不同程度的F10表达下调,其中2个克隆F10基因表达下调超过70%,其中一个克隆下调达到74%,确定为F10有效沉默的阳性克隆。三小结1、对F10基因设计能转录生成发卡样siRNA的寡核苷酸片段,构建出pRetroQ-iF10重组体。对构建的pRetroQ/i F10重组质粒测序结果证实shRNA表达模板成功克隆于pRetroQ载体上。2、用设计分析软件进行筛选序列,确定能制备针对F10基因siRNA的寡核苷酸序列。合成双链DNA,连接到逆转录病毒载体pRetroQ上构建重组质粒pRetroQ-iF10,脂质体转染KLE细胞,嘌呤霉素筛选,获得单细胞克隆。3、对筛选获得的单细胞克隆进行F10基因的荧光定量RT-PCR检测并确定了F10有效沉默的阳性克隆。第三部分F10基因沉默对KLE细胞凋亡的影响及机理一研究内容与方法以F10基因表达稳定下调的KLE细胞克隆为模型,深入探讨F10基因沉默对KLE细胞凋亡的影响及机制。Annexin-v检测KLE细胞凋亡,荧光定量RT-PCR和western blot检测细胞色素C和Bcl-2表达水平。二结果1、流式细胞术检测发现:阴性对照组细胞的凋亡率为1.8%,F10基因沉默的细胞克隆,其凋亡率增加至16.8%,凋亡水平显著高于对照组。2、荧光定量PCR检测显示,F10沉默的细胞克隆中细胞色素C mRNA表达升高达到85%,显著高于对照组(P＜0.05)KLE细胞与对照组细胞之间无显著差异。3、Western印迹检测结果显示:KLE细胞、阴性对照组细胞和F10沉默实验组细胞线粒体中细胞色素C蛋白的相对表达量分别为:0.46、0.38和0.13,F10沉默实验组呈现蛋白表达降低。而胞浆中细胞色素C蛋白的相对表达量分别0.15、0.15和0.44,表现为F10沉默实验组中细胞色素C蛋白的相对表达量显著升高。Bcl-2 mRNA蛋白的相对表达量分别为:0.40、0.41和0.11。从KLE细胞的0.4到F10沉默实验组的0.11,F10沉默实验组细胞克隆中Bcl-2表达下调达64%。三小结以F10基因表达稳定下调的KLE细胞克隆为模型,深入探讨F10基因沉默对KLE细胞凋亡的影响及机制。1、F10沉默的细胞克隆中BCL-2表达下调,说明线粒体途径参与了F10基因沉默诱导的细胞凋亡的机制。2、F10基因沉默诱导细胞凋亡的可能机制:细胞色素C的释放是F10沉默诱导KLE细胞凋亡的途径之一。全文结论1、体外转录制备针对F10基因的dsRNA能特异有效沉默KLE细胞中的F10基因。2、F10基因的沉默可诱导KLE细胞的凋亡。3、通过在F10基因沉默载体基础上,成功建立了F10基因表达下调的稳定细胞克隆4、F10沉默的细胞克隆中BCL-2表达下调,提示线粒体途径参与了F10基因沉默诱导的细胞凋亡的机制。5、细胞色素C的释放提示是F10沉默诱导KLE细胞凋亡的途径之一。更多还原

【Abstract】 Gestational trophoblastic diseases include complete,partial and invasive moles, placental site trophoblastic tumours and choriocarcinomas,occurrence and development of GTD involves multifactorial pathological processes with multiple genetic alterations including activation of oncogenes and inactivation of tumour suppressor genes.Because gestational trophoblasts are special cells which are different from other somatic cells,they have specific biological and differentiational characteristics.Therefore it becomes very important to study the mechanism of GTD evolution and to search for novel effective methods of diagnosis and therapy.RNA interference was initially discovered in C.elegans(roundworm).RNAi is a sequence-specific posttranscriptional gene silencing mechanism,triggered by double-stranded RNA(dsRNA) and causes degradation of mRNAs homologous in sequence to the dsRNA.In 2001,Tuschl and his colleagues initially reported that chemically synthesized 21 nt siRNA(small interfering RNA)duplexes specifically suppress the expression of endogenous and heterologeous genes in different mammalian cell lines,and no non-specific gene silencing effects were observed.It is now well known that RNAi has become a powerful tool for studying gene function. Antisense oligonucleotide and ribozyme technologies,have been useful in reverse genetics,but only to a limited degree.At present,reverse genetics is the most effective way to assess the function of a gene,but so far there has been no general method for reverse genetics other than gene targeting by homologous recombination, which is costly and slow.Techniques such as antisense oligonucleotide and ribozyme technologies,have been useful in reverse genetics,but only to a limited degree.RNAi has been widely used to study gene function owing the following advantages: (ⅰ) DsRNA is much more stable than single-stranded RNA and more resistant to be degraded;(ⅱ)RNAi is a potent method,requiring only a few molecules of dsRNA per cell to silence gene expression;(ⅲ)High sequence specificity—Only one nucleotide mutation in the target mRNA will make the dsRNA ineffective;(ⅳ) Most of the genes in the genome of various organisms can be knocked down by RNAi. Therefore,RNA interference has attracted the attention of many researchers.The overexpression of oncogenes are genomic features of human cancers.Inhibition of abnormal expression of these oncogenes has been a common idea for studying the function of F10 gene.In 2005,Suppressive subtractive hybridization method was applied.Subtractive cDNA library was created between hydatidiform mole and normal early-pregnancy villous tissues.New genes were screened and full-length sequence were cloned.Totally 28 clones were selected from the cDNA library,10 gene sequences were identified,and 3 genes were found to be new genes.The full length sequence of one gene—F10 was cloned.The aim was to show that F10 gene might be closely associated with the pathogenesis of hydatidiform mole.Hybridization was used to study the expression of F10 in 12 cases of hydatidiform mole,6 cases of invasive mole,and 8 cases of choriocarcinoma.F10 mRNA was positive in all cases of hydatidiform mole,invasive mole,and choriocarcinoma,and the expression intensity significantly increased in ascending order with hydatidiform mole,invasive mole and choriocarcinoma.Analysis of results suggested that the expression of F10 gene may relate to the occurrence and invasiveness of trophoblastic tumor,with possible involvement in the invasion or malignant changes of trophoblastic cells.In 2006,In situ hybridization was used to study the expression of F10mRNA in different tumor tissues and normal tissues.F10 mRNA was shown to be positive in adenocarcinoma of ovarian carcinoma, endometrium carcinoma,mammary carcinoma,hepatocarcinoma and gastric carcinoma.There was no significant difference at the expression intensity of different adenocarcinomas.F10 mRNA was negative in normal tissues and hepatocarcinoma para-tumor tissues.Higher expression of F10 gene may be closely associated with trophoblastic tumors,suggesting an important role in the development of the adenocarcinoma.A novel related gene F10 expressed highly in HM was detected and subtractive cDNA library between normal villus,early pregnancy,choriocarcinoma and HM tissues was achieved in our study using suppression subtractive hybridization technique.An investigation of the features of F10 expression and essential functions were carried out with analysis of the gene silencing technique based on RNA interference in this study.Chapter 1 Knock-down of F10 expression by RNAi induces apoptosis of KLE cellContents and MethodsTo explore the small member a chain RNA to the KLE cell the silent effect of the F10 genes,and confirm the influence of F10 gene silencing on KLE cell apoptosis;The dsRNA aim to F10 genes was produced by in vitro transcription;plasmid was transfected into KLE cells.The expression level of F10 gene was detected by fluorescence quantitative RT-PCR in different time after KLE cell was transfected. The difference of apoptosis of KLE cell was assayed by FCM(flow cytometry) after F10 gene silencing.Results1、Sufficient dsRNA can be prepared by in vitro transcription.Molecular weight of dsRNA is about 21 bp by crylicacyl-amine gel electrophoresis,and the concentration of dsRNA is 740 ng/μl(52.85μmol/L).2 The results by fluorescence quantitative RT-PCR of the Taqman probe method indicate that compared with the control group,the copy count of F10 gene decreases after the KLE cells were transfected by dsRNA with different concentration,including 10nM,20nM and 50nM,and the decrease is remarkable when using dsRNA with 20nM and 50nM.The phenomena had been maintained till 72 hours after transfection.Among these nanomolars,the most significant effect of F10 gene silencing happened in KLE cell transfected with 20 nM dsRNA for 48h,the expression of F10 mRNA down regulated to 83%.3 The DNA content in KLE is detected by FCM(flow cytometry) and the results show that when compared with control group,the apoptosis index of KLE cells transfected with dsRNA increased from 0.36%to 8.91%,which is 24.75 times the control.Summary1、The dsRNA aim to F10 genes was produced by in vitro transcription and plasmid transfected into KLE cells.Expression level of F10 gene was detected by fluorescence quantitative RT-PCR in different time after KLE cell was transfected.The result to show that F10 gene in KLE cells can be specifically knocked-down with dsRNA produced by in vitro transcription.2、The DNA content in KLE is detected by FCM(flow cytometry) and the results show that the down regulation of F10 gene induces apoptosis of KLE cells. Chapter 2 The establishment of silent vector and setting up the stable cloneContents and MethodsDesign siRNA oligonucleotide snippet with hair card kind aim at the F10 gene. Set up pRetroQ-i vector aim at the F10 and establish the down -regulation of KLE cell line under the F10 gene expression stability.Depending on the F10 gene sequence in GenBank(Genbank NO.gi:5641046),design the candidate sequence by the soft and confirm the siRNA sequence aim to F10 gene.Synthesize siRNA oligonucleotide with hair card kind.The recombinant plasmid pRetroQ-i F10 producing short hairpin RNA of F10 gene was constructed by legating the double DNA annealed with two synthesizing oligonucleotides to pRetroQ,pRetroQ-i F10 plasmid was transfected into KLE cells,then the cells were screened by puromycine, and a single clone was established.Results1 The sequencing is correct and the established F10 plasmid showed that shRNA expression template successfully cloned to the pRetroQ/i F10 vector.The plasmid with the right insert sequences was chosen to make the single clone.2 The detection of F10 and GAPDH gene in single clone by fluorescence quantitative RT-PCR displayed that the expression level of F10 was not significantly differente between two RetrQ/negative cell clones,while it had down-regulated with different degrees among the five pRetrQ/RNAi.The expression levels of F10 descended by over 70%in two clones,and in the No.5 clone it descended to adjust to 74%.It is doubtlessly the effective F10 knocking-down positive clone.Summary1、Design siRNA oligonucleotide snippet with hair card kind aim at the F10 gene.Set up pRetroQ-i vector aim at the F10.The results sequencing is correct and the established F10 plasmid showed that shRNA expression template successfully cloned to the pRetroQ/i F10 vector.2、Depending on the F10 gene sequence in GenBank(Genbank NO.gi:5641046), design the candidate sequence by the soft and confirm the siRNA sequence aim to F10 gene.Synthesize siRNA oligonucleotide with hair card kind.The recombinant plasmid pRetroQ-i F10 producing short hairpin RNA of F10 gene was constructed by legating the double DNA annealed with two synthesizing oligonucleotides to pRetroQ.pRetroQ-i F10 plasmid was transfected into KLE cells,then the cells were screened by puromycine,and single clone was established.3 The detection of F10 gene in single clone by fluorescence quantitative RT-PCR displayed that the No.5 clone it down regulated to adjust to 74%.It is doubtlessly the effective F10 knocking-down positive clone.Chapter 3 Effect and mechanism of KLE cell aapoptosis induced by silencing F10 geneContents and MethodsTo explore the effect and mechanism of F10 silencing on apoptosis of KLE by setting up a cell line in which F10 gene is knocked-down..The apoptosis of KLE cell was detected by FCM(flow cytometry).and Fluorescence quantitative RT-PCR together with western blot were used to examine the expression of cytochrome and Bcl-2 mRNAResults1 The apoptosis of KLE cell by FCM displays that the rate of cell apoptosis from negatively matched control is 1.8%,while in cell clone of F10 knocked-down gene,it increases to 16.8%,and is distinctly higher than the matched control.2 The results form fluorescence quantitative RT-PCR show that the expression of cytochrome in F10 knocked-down gene cell clone increases to 85%,while that of KLE cell has no significant difference compared to the matched control.3 The results from Weston blot detection show that the relative expression of cytochrome C is 0.46,0.38 and 0.13,corresponding to in KLE cell,control group cell and in mitochondrion of experimental group.The relative expression of cytochrome C in cytoplasm is 0.15,0.15 and 0.44 respectively,which is significantly higher in the group of disturbed F10 gene.The expression of bcl-2 m-RNA was significantly down-regulated to 64%,and the relative expression of protein descended to 0.11 from 0.44,which is the control group.SummaryExplore the effect and mechanism of F10 silencing on apoptosis of KLE by setting up a cell line in which F10 gene is knocked-down.1、The cell clone of knocked-down F10 gene induced decreased expression of BCL-2 illuminating mitochondria participated in the mechanism of F10 gene silencing effect on cell apoptosis.2、The release of cytochrome C may be one of the pathways by which F10 gene knocking-down induced KLE cell apoptosis.Conclusion1 F10 gene in KLE cells can be specifically knocked-down with dsRNA produced by in vitro transcription2 The silencing of F10 gene induces the apoptosis of KLE cells.3 The expression of F10 is stably down-regulated By building a cell clone with F10 gene silencing and this is the bases of the further experiment.4 The cell clone of F10 gene knocking-down induced decreased in expression of BCL-2 and illuminated mitochondria have participated in the mechanism of F10 gene silencing effect on cell apoptosis.5 the release of cytochrome C may be one of the pathways by which F10 gene knocking-down induced KLE cell apoptosis.更多还原