脱氧核酶抑制突变型p53基因表达的体外研究

发布时间：2018-07-13 11:42

【摘要】：背景与目的肿瘤的发生是由于某些原癌基因的激活、抑癌基因的失活以及凋亡相关基因的改变导致细胞增殖、分化和凋亡失调的结果。作为“基因组卫士”(guardian of genome)的 p53 基因,是迄今发现与人类肿瘤相关性最为密切的一个抑癌基因,人类 50%以上的肿瘤可检测出 p53 基因的突变。突变型 p53(mp53)不仅能以“显性负效应”阻碍野生型 p53(wp53)的抑癌功能,而且还能“重获新功能”,具有异常的转录激活、降低基因组 DNA 稳定性、抑制细胞分化、刺激肿瘤细胞无限增殖等癌基因的活性。转染 wp53 基因治疗肿瘤,在体外研究中已显示明显的抑癌效应,但临床研究效果却不理想。近年,国内外已有用反义寡聚核苷酸(ASO)、核酶(ribozyme, RZ)阻止或修复 mp53 基因表达的报道。脱氧核酶(DNAzyme, DZ)是一种崭新的、能够抑制细胞内 RNA 表达的核酸分子。它是通过对体外合成的随机序列进行筛选获得的、具有 RNA 切割功能的 DNA 分子。其中, -23”型脱氧核酶(“10-23”DZ) “10功能最强,此酶由一个 15 个核苷酸(nt)的催化中心和与靶 RNA 碱基互补的两个侧翼序列构成,能在靶 RNA 未配对的嘌呤和配对的嘧啶残基之间发生序列特异性切割效应。目前,“10-23”DZ 的应用研究在病毒感染性疾病、肿瘤、心血管疾病等方面已取得了不同程度的进展,但尚没有脱氧核酶抑制突变型 p53 基因表达的研究。本研究针对突变型 p53(R273H) mRNA 设计三种脱氧核酶,观察其在无细胞体系切割 mp53 mRNA 的有效性和特异性,体外筛选出能有效切割 mp53 mRNA,对 wp53 mRNA 无切割或切割效率不高的 DZ。 4 观察其在HT29结肠癌细胞内抑制mp53 mRNA和蛋白质表达的效应及对 HT29 细胞的生长抑制作用,探索脱氧核酶在肿瘤 p53 基因治疗的可能性。方法 1. 设计合成 DZ:根据 p53 突变数据库的信息以及文献报道的人类肿瘤 p53 基因突变的特征,结合“10-23”DZ 切割 RNA 特点,用 RNAstructure 及 RnaViz 软件分析 p53 mRNA 的二级结构,综合分析并设计合成针对 mp53 的“10-23”DZ。 2. 无细胞系统观察 DZ 切割效应:RT-PCR 分别扩增 HT29 细胞的 mp53 和 A549 细胞的 wp53 的 cDNA 片段(345bp),将其定向克隆到 pBluescript II KS(+)噬菌体 T7 启动子的下游,获得重组质粒 mp53pBs 和 wp53pBs,经体外转录分别获得 392bp 的突变型和野生型 p53 基因的单链 RNA 片段,作为 DZ 切割反应的底物。在适当镁离子浓度、温度、PH 值的条件下观察所设计的“10-23”DZ 对底物的切割效应。 3. 细胞内观察 DZ 效应:经脂质体或胆固醇将筛选出的 DZ 或 ASO 转染入 HT29 结肠癌细胞株,RT-PCR 检测 mp53 mRNA 水平,观察各种DZ及ASO对HT29细胞mp53 mRNA的影响,免疫细胞化学及Image Pro Plus 4.5图像分析系统检测mp53蛋白,观察各种DZ及ASO对mp53 蛋白表达的影响。 MTT 法初步评估 DZ 抑制 HT29 细胞生长效应。结果 1. DZ 的设计合成:综合各种因素设计合成针对 mp53 (R273H, CGTCAT),在突变点之后第二、三个碱基之间发生切割、不同臂长的三种“10-23”DZ:p53DZ7、p53DZ9、p53DZ11 及与 p53DZ11 相同靶位的反义对照 p53ASO 和 DZ 突变体对照 mutp53DZ。为了增加 DZ 细胞内稳定性及细胞吸收率,结合体外筛选结果合成硫代和/或胆固醇修饰
[Abstract]:Background and purpose
The occurrence of tumor is due to the activation of some proto oncogenes, the inactivation of tumor suppressor genes and the changes of apoptosis related genes that result in cell proliferation, differentiation and apoptosis disorder. As the p53 gene of "guardian of genome", it is one of the most closely related tumor suppressor genes that have been found to be associated with human swelling to date, human 5 More than 0% of the tumor can detect the mutation of the p53 gene. The mutant p53 (mP53) can not only obstruct the tumor suppressor function of the wild type p53 (wp53) with "dominant negative effect", but can "regain the new function", have abnormal transcriptional activation, reduce the stability of genomic DNA, inhibit the differentiation of the cell and stimulate the tumor cells to proliferate indefinitely and so on. The effect of transfection of wp53 gene in the treatment of tumor has shown obvious tumor suppressor effect in the study in vitro, but the effect of clinical research is not ideal. In recent years, the reports of antisense oligonucleotides (ASO) and ribozyme (RZ) have been used to prevent or repair the expression of mP53 gene.
DNAzyme (DZ) is a new type of nucleic acid molecule that inhibits the expression of RNA in cells. It is a DNA molecule with RNA cutting function by screening random sequences synthesized in vitro. Among them, -23 "type deoxy ribozyme (" 10-23 "DZ)" has the strongest 10 function, the enzyme is stimulated by a 15 nucleotide (NT). " The chemical center and the two flanking sequences complementing the target RNA base are composed of a sequence specific cutting effect between the unpaired purines and the paired pyrimidine residues of the target RNA. At present, the application of "10-23" DZ has been progressed in different degrees in viral infectious diseases, tumors, and cardiovascular diseases. Deoxy ribozyme inhibits mutant p53 gene expression.
In this study, three deoxy ribozymes were designed for mutant p53 (R273H) mRNA, and the effectiveness and specificity of mP53 mRNA in cell free system were observed. In vitro, mP53 mRNA was effectively cut, wp53 mRNA was not cut or the cutting efficiency was not high in DZ..
Four
To observe its inhibitory effect on mP53 mRNA and protein expression in HT29 colon cancer cells.
To inhibit the growth of HT29 cells, explore the feasibility of deoxy ribozyme in tumor p53 gene therapy.
Ability.
Method
1. design and synthesize DZ: based on p53 mutation database and human being reported in literature.
The characteristics of tumor p53 gene mutation are combined with the characteristics of "10-23" DZ cutting RNA.
RNAstructure and RnaViz software are used to analyze the two level structure of p53 mRNA.
Design and synthesize "10-23" DZ. for mP53
2. no cell system was used to observe DZ cleavage effect: RT-PCR amplifying HT29 cells respectively.
The cDNA fragment (345bp) of wp53 from mP53 and A549 cells was cloned.
PBluescript II KS (+) phage T7 promoter was downstream, and the recombinant plasmid mp53pBs was obtained.
Wp53pBs and 392bp were obtained from in vitro transcriptional and wild type p53 genes.
Single strand RNA fragments are used as substrates for DZ cleavage reactions.
The effect of the "10-23" DZ on the substrate was observed under the conditions of pH and pH.
3. the DZ effect was observed in cells: DZ or ASO screened by liposomes or cholesterol.
Transfected into HT29 colon cancer cell line, RT-PCR was used to detect mP53 mRNA level.
Effects of DZ and ASO on mP53 mRNA in HT29 cells, immunocytochemistry and Image
Pro Plus 4.5 image analysis system detects mP53 protein, observing all kinds of DZ and ASO to mP53.
MTT assay was used to evaluate the inhibitory effect of DZ on the growth of HT29 cells.
Result
1. the design and synthesis of DZ: combining various factors to design and synthesize mP53 (R273H).
CGTCAT) cuts between second, third bases after the mutation point, with different arm lengths.
Three "10-23" DZ:p53DZ7, p53DZ9, p53DZ11 and the same target as p53DZ11.
The antisense p53ASO and DZ mutants contrast mutp53DZ. in order to increase DZ cells.
Internal stability and cell absorptivity, combined with in vitro screening results, to synthesize thiol and / or cholesterol modifiers.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

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