【作者】 吕佳音；

【导师】 高忠礼；

【作者基本信息】 吉林大学 ， 外科学， 2009， 博士

【摘要】 目的观察靶向MDM2的小干扰RNA（siRNA）对骨肉瘤生长的影响。方法1、应用免疫组化方法检测MDM2在人骨肉瘤组织的表达;2、以MDM2已知序列为靶点,构建PGCsilencer^TM-MDM2 siRNA重组质粒;3、通过体外、体内实验,研究重组质粒转染骨肉瘤U20细胞及裸鼠瘤内注射重组质粒后MDM2的表达情况及细胞凋亡情况。结果1、免疫组织化学研究结果提示MDM2的过度表达在骨肉瘤的发生、发展过程中起重要作用。2、成功地构建了PGCsilencer^TM-MDM2 siRNA重组质粒,为进一步研究MDM2结构与功能及RNAi技术的应用提供了方便条件。3、成功地将重组质粒转染入骨肉瘤U20细胞,为后续的研究工作作好了铺垫。4、PGCsilencer^TM-MDM2 siRNA重组质粒转染U20骨肉瘤细胞后MDM2的表达无论在蛋白水平还是在基因水平上都明显下调,体外研究结果证实,RNA干涉MDM2基因的效果具有特异性和高效性。5、成功地建立了骨肉瘤裸鼠动物模型:通过体内实验,证实瘤内注射PGCsilencer^TM-MDM2 siRNA重组质粒且应用电穿孔技术可明显抑制裸鼠骨肉瘤的生长,降低肿瘤体积及重量。结果显示,MDM2的表达明显受抑,且促进了细胞的凋亡及肿瘤组织坏死。结论应用RNAi技术沉默MDM2基因可以降低骨肉瘤细胞中MDM2的表达,导致细胞凋亡进而抑制肿瘤的生长。更多还原

【Abstract】 Osteosarcoma is the most common type of malignant bone cancer in the young people,and deeply threatens the health and life quality.Therefore it becomes a very important region in studying the mechanism of malignant cancer evolution,searching the novel effective methods of diagnosis and therapy and clerifying the theory basis of improving prognosis,with the completion of large-scale genome sequencing,thousands of genes have been identified,and we know nothing about their function.The overexpression of oncogenes are genomic features of human cancers.Inhihition of abnormal expression of there oncogenes has been a commom idea for development of the malignant therapy.Some research has been testified that MDM2 existed in osteosarcoma and signal conduction path was related with cell’s proliferation.It has been identified that RNA interference signaling techology is amore powerful tool.Investigate the inhibitory effect of the small interfering RNA targeting on MDM2 gene,on the growth of osteosarcoma in this study.The human homologue of the murine double minute 2 gene (MDM2),a p53-binding protein which may act as a regulator of p53 protein function,has recently been cloned.Initial studies of this gene in a variety of human tumors have shown frequent gene amplification in most types of sarcomas, including osteosarcomas.Amplification of the MDM2 gene may produce a functional inactivation of the p53 protein.To examine possible clinical or pathological correlates of MDM2 gene amplification in osteosarcoma,MDM2 gene amplification may be associated with tumor progression and metastasis in osteosarcoma.Further investigation is warranted on the potential clinicopathological correlates of MDM2 gene amplification in osteosarcoma.The p53 gene is the most frequently altered gene in human malignancies and has also been implicated in the development of a number of companion animal tumours.One mechanism by which mdm2 may down regulate p53,is to target p53 for degradation.The p53 protein is maintained in normal cells as an unstable protein,and its interaction with p53 can target p53 for degradation via a ubiquitin proteosome pathway.MDM2 can also control p53 function by suppressing p53 transcriptional activity.MDM2 is a transcriptional target of p53 and expression is induced by the binding of p53 to an internal promoter within the MDM2 gene.MDM2 can in turn bind to a domain within the amino terminus of p53 thereby inhibiting the transcriptional activity and G1 arrest function of p53 by masking access to the transcriptional machinery.Gene amplification of MDM2 has been found almost exclusively in human sarcomas and suggests that this is an alternative mechanism for inactivating p53 in these tumours.The p53 protein is maintained in normal cells as an unstable protein,and its interaction with p53 can target p53 for degradation via a ubiquitin proteosome pathway.MDM2 can also control p53 function by suppressing p53 transcriptional activity.MDM2 is a transcriptional target of p53 and expression is induced by the binding of p53 to an internal promoter within the mdm2 gene.Small interfering RNA(SiRNA),sometimes known as short interfering RNA or silencing RNA.Most notably,siRNA is involved in the RNA interference(RNAi) pathway,where it interferes with the expression of a specific gene.In addition to their role in the RNAi pathway,siRNAs also act in RNAi-related pathways,e.g.,as an antiviral mechanism or in shaping the chromatin structure of a genome;the complexity of these pathways is only now being elucidated. Given the ability to knock down essentially any gene of interest,RNAi via siRNAs has generated a great deal of interest in both basic and applied biology.There are an increasing number of large-scale RNAi screens that are designed to identify the important genes in various biological pathways.Because disease processes also depend on the activity of multiple genes,it is expected that in some situations turning off the activity of a gene with an siRNA could produce a therapeutic benefitInvestigate the inhibitory effect of the small interfering RNA targeting on MDM2 gene,on the growth of osteosarcoma.PGCsilencerTM-MDM2 siRNA was constructed and transfected into the osteosarcoma cell line U20S cell.The inhibition effects on MDM2 were determined by RT-PCR and Western blot analysis.The cellular growth activities were determined by MTT assay,and the cell apoptosis was examined by flow cytometry.The therapeutic effects of siMDM2 was assessed on the nude mouse model of transplanted tumor.The siMDM2 plasmid was successfully constructed.After siMDM2 being transfected into the U20S cells,the expressions of MDM2 gene and protein were significantly inhibited.The ability of cell growth activities decreased greatly and the cell apoptosis occurred apparently.There was no significant difference between the negative,controlled group and non-transfected group.The growth of xenograft tumor in siMDM2 transfected nude mice was inhibited and the expressions of MDM2 gene and protein were down-regulated remarkably.SiRNA targeting MDM2 gene inhibits the MDM2 expression in osteosarcoma U20S cells and the growth of osteosarcoma in nude mice.In summary,we successfully constructed PGCsilencer TM-MDM2 siRNA plasmid.SiRNA of MDM2 suppressed MDM2 expression in U20 cells,inhibited tumor cells proliferation and increased cell apoptosis significantly.This study demonstrated that in vivo delivery of siRNA into tumor mass results in effective inhibition of expression of gene encoding MDM2 and suppressed growth and induced apoptosis of the cancer cells.MDM2 decresae and wt p53 increase may be provide theory foundation for cancer’s prognosis and treatment.Furthermore, RNAi rechnique may be a promising tool for osteosarcoma therapy.更多还原