【摘要】：背景與目的 腫瘤的發(fā)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關(guān)基因的改變導(dǎo)致細(xì)胞增殖、分化和凋亡失調(diào)的結(jié)果。作為“基因組衛(wèi)士”(guardian of genome)的 p53 基因,是迄今發(fā)現(xiàn)與人類腫瘤相關(guān)性最為密切的一個抑癌基因,人類 50%以上的腫瘤可檢測出 p53 基因的突變。突變型 p53(mp53)不僅能以“顯性負(fù)效應(yīng)”阻礙野生型 p53(wp53)的抑癌功能,而且還能“重獲新功能”,具有異常的轉(zhuǎn)錄激活、降低基因組 DNA 穩(wěn)定性、抑制細(xì)胞分化、刺激腫瘤細(xì)胞無限增殖等癌基因的活性。轉(zhuǎn)染 wp53 基因治療腫瘤,在體外研究中已顯示明顯的抑癌效應(yīng),但臨床研究效果卻不理想。近年,國內(nèi)外已有用反義寡聚核苷酸(ASO)、核酶(ribozyme, RZ)阻止或修復(fù) mp53 基因表達(dá)的報道。 脫氧核酶(DNAzyme, DZ)是一種嶄新的、能夠抑制細(xì)胞內(nèi) RNA 表達(dá)的核酸分子。它是通過對體外合成的隨機(jī)序列進(jìn)行篩選獲得的、具有 RNA 切割功能的 DNA 分子。其中, -23”型脫氧核酶(“10-23”DZ) “10功能最強(qiáng),此酶由一個 15 個核苷酸(nt)的催化中心和與靶 RNA 堿基互補(bǔ)的兩個側(cè)翼序列構(gòu)成,能在靶 RNA 未配對的嘌呤和配對的嘧啶殘基之間發(fā)生序列特異性切割效應(yīng)。目前,“10-23”DZ 的應(yīng)用研究在病毒感染性疾病、腫瘤、心血管疾病等方面已取得了不同程度的進(jìn)展,但尚沒有脫氧核酶抑制突變型 p53 基因表達(dá)的研究。 本研究針對突變型 p53(R273H) mRNA 設(shè)計三種脫氧核酶,觀察其在無細(xì)胞體系切割 mp53 mRNA 的有效性和特異性,體外篩選出能有效切割 mp53 mRNA,對 wp53 mRNA 無切割或切割效率不高的 DZ。 4 觀察其在HT29結(jié)腸癌細(xì)胞內(nèi)抑制mp53 mRNA和蛋白質(zhì)表達(dá)的效應(yīng)及 對 HT29 細(xì)胞的生長抑制作用,探索脫氧核酶在腫瘤 p53 基因治療的可 能性。 方 法 1. 設(shè)計合成 DZ:根據(jù) p53 突變數(shù)據(jù)庫的信息以及文獻(xiàn)報道的人類 腫瘤 p53 基因突變的特征,結(jié)合“10-23”DZ 切割 RNA 特點(diǎn),用 RNAstructure 及 RnaViz 軟件分析 p53 mRNA 的二級結(jié)構(gòu),綜合分析并 設(shè)計合成針對 mp53 的“10-23”DZ。 2. 無細(xì)胞系統(tǒng)觀察 DZ 切割效應(yīng):RT-PCR 分別擴(kuò)增 HT29 細(xì)胞的 mp53 和 A549 細(xì)胞的 wp53 的 cDNA 片段(345bp),將其定向克隆到 pBluescript II KS(+)噬菌體 T7 啟動子的下游,獲得重組質(zhì)粒 mp53pBs 和 wp53pBs,經(jīng)體外轉(zhuǎn)錄分別獲得 392bp 的突變型和野生型 p53 基因 的單鏈 RNA 片段,作為 DZ 切割反應(yīng)的底物。在適當(dāng)鎂離子濃度、溫 度、PH 值的條件下觀察所設(shè)計的“10-23”DZ 對底物的切割效應(yīng)。 3. 細(xì)胞內(nèi)觀察 DZ 效應(yīng):經(jīng)脂質(zhì)體或膽固醇將篩選出的 DZ 或 ASO 轉(zhuǎn)染入 HT29 結(jié)腸癌細(xì)胞株,RT-PCR 檢測 mp53 mRNA 水平,觀察各 種DZ及ASO對HT29細(xì)胞mp53 mRNA的影響,免疫細(xì)胞化學(xué)及Image Pro Plus 4.5圖像分析系統(tǒng)檢測mp53蛋白,觀察各種DZ及ASO對mp53 蛋白表達(dá)的影響。 MTT 法初步評估 DZ 抑制 HT29 細(xì)胞生長效應(yīng)。 結(jié) 果 1. DZ 的設(shè)計合成:綜合各種因素設(shè)計合成針對 mp53 (R273H, CGTCAT),在突變點(diǎn)之后第二、三個堿基之間發(fā)生切割、不同臂長的 三種“10-23”DZ:p53DZ7、p53DZ9、p53DZ11 及與 p53DZ11 相同靶位 的反義對照 p53ASO 和 DZ 突變體對照 mutp53DZ。為了增加 DZ 細(xì)胞 內(nèi)穩(wěn)定性及細(xì)胞吸收率,結(jié)合體外篩選結(jié)果合成硫代和/或膽固醇修飾
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R346

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