本文選題：白血病 + HOXA5��； 參考：《濱州醫(yī)學(xué)院》2015年碩士論文

【摘要】：目的：同源盒基因(homeobox gene,HOX)是一組在進(jìn)化過程中高度保守的基因,通過編碼重要的轉(zhuǎn)錄因子特異性結(jié)合、調(diào)控靶基因,在胚胎早期發(fā)育和造血細(xì)胞增殖、分化、凋亡等過程中發(fā)揮重要的調(diào)控作用。HOXA5是HOX家族的一員,定位于7號(hào)染色體,其編碼的DNA結(jié)合轉(zhuǎn)錄因子,在調(diào)節(jié)基因的表達(dá)、細(xì)胞的分化和機(jī)體形態(tài)的發(fā)生中發(fā)揮重要作用。本研究采用RNAi技術(shù),構(gòu)建并篩選有效干擾HOXA5基因表達(dá)的重組質(zhì)粒載體pGPU6/GFP/Neo-HOXA5 shRNA,觀察HOXA5基因表達(dá)抑制后對(duì)人白血病細(xì)胞株U937增殖、凋亡的影響。方法：1.靶向設(shè)計(jì)合成3對(duì)針對(duì)HOXA5基因不同位點(diǎn)的siRNA序列,構(gòu)建構(gòu)建3種pGPU6/GFP/Neo-HOXA5 shRNA-1,-2,-3重組質(zhì)粒載體并進(jìn)行測(cè)序及鑒定;載體轉(zhuǎn)染人白血病細(xì)胞株U93748 h后,熒光定量PCR、Western blotting法檢測(cè)HOXA5 mRNA及蛋白表達(dá),篩選出沉默HOXA5效果最佳的重組質(zhì)粒載體。2.將已篩選出的HOXA5 shRNA重組質(zhì)粒載體轉(zhuǎn)染U937細(xì)胞,CCK-8法和流式細(xì)胞術(shù)檢測(cè)pGPU6/GFP/Neo-HOXA5 shRNA對(duì)U937細(xì)胞增殖能力和細(xì)胞周期、細(xì)胞凋亡的影響,Western blotting檢測(cè)細(xì)胞凋亡相關(guān)蛋白Survivin, Caspase-3的表達(dá)變化。結(jié)果：1.成功構(gòu)建3種pGPU6/GFP/Neo-HOXA5重組質(zhì)粒載體,轉(zhuǎn)染48后熒光定量RT-PCR及Western blotting分析檢測(cè)發(fā)現(xiàn)3種重組質(zhì)粒載體均能抑制HOXA5的表達(dá),其中shRNA3的沉默效率最高。2.將篩選出的重組質(zhì)粒載體轉(zhuǎn)染U937細(xì)胞,Western blotting和熒光定量RT-PCR法檢測(cè)結(jié)果顯示,相較于空白對(duì)照組和陰性對(duì)照組相比,轉(zhuǎn)染pGPU6/GFP/Neo-HOXA5 shRNA-3重組質(zhì)粒載體的U937細(xì)胞能明顯抑制HOXA5基因的表達(dá),且沉默效率可達(dá)70%(P0.05)。CCK-8法檢測(cè)發(fā)現(xiàn)分別轉(zhuǎn)染U937細(xì)胞0,24,48,72 h, HOXA5 shRNA能明顯抑制U937細(xì)胞增殖,且生長(zhǎng)抑制率隨轉(zhuǎn)染時(shí)間的延長(zhǎng)逐漸增加(P0.05)。流式細(xì)胞術(shù)檢測(cè)顯示實(shí)驗(yàn)組G0/G1期細(xì)胞數(shù)明顯多于空白對(duì)照組和陰性對(duì)照組,S期細(xì)胞數(shù)則減少。Annexin V-FITC雙染法檢測(cè)結(jié)果發(fā)現(xiàn),空白對(duì)照組、陰性對(duì)照組、HOXA5shRNA組的細(xì)胞凋亡率分別為(7.36±1.63)%、(8.10±1.39)%、(26.52±2.35)%, HOXA5 shRNA組的細(xì)胞凋亡率明顯高于其余兩組(P0.05)。Western blotting檢測(cè)顯示：HOXA5 shRNA干擾U937細(xì)胞48 h后,HOXA5 shRNA組Survivin蛋白的表達(dá)量明顯低于陰性對(duì)照組和空白對(duì)照組,而Caspase-3蛋白的表達(dá)量明顯高于其余兩組(P0.05)。結(jié)論：1.成功構(gòu)建了靶向沉默HOXA5的重組質(zhì)粒表達(dá)載體pGPU6/GFP/Neo-HOXA5 shRNA-1,-2,-3,且篩選出沉默HOXA5效果最佳的重組質(zhì)粒載體。2.重組表達(dá)載體pGPU6/GFP/Neo-HOXA5-3,轉(zhuǎn)染U937細(xì)胞后能有效沉默HOXA5基因,抑制HOXA5 mRNA和蛋白的表達(dá)：RNA干擾HOXA5基因的表達(dá)能顯著抑制U937細(xì)胞增殖,誘導(dǎo)周期阻滯,促進(jìn)凋亡,后者可能與RNA干擾引起抑制凋亡基因Survivin蛋白表達(dá)量減少、Caspase-3蛋白表達(dá)量增多相關(guān)。
[Abstract]:Objective: homeobox gene (HOX) is a group of highly conserved genes in the process of evolution. By coding important transcription factor specific binding and regulating target genes,.HOXA5 plays an important role in the process of early embryonic development and hematopoietic cell proliferation, differentiation and apoptosis..HOXA5 is a member of the HOX family and is located in No. 7 staining. In vivo, its encoded DNA combined with transcription factors play an important role in regulating gene expression, cell differentiation and the occurrence of body morphology. This study uses RNAi technology to construct and screen the recombinant plasmid vector pGPU6/GFP/Neo-HOXA5 shRNA that effectively interferes with the expression of HOXA5 gene, and observes the inhibition of HOXA5 gene expression to human leukemia cell line U9 37 the effect of proliferation and apoptosis. Method: 1. target design and synthesis of 3 pairs of siRNA sequences against HOXA5 gene, construction and construction of 3 pGPU6/GFP/Neo-HOXA5 shRNA-1, -2, -3 recombinant plasmid carrier and sequencing and identification; after transfection of human leukemia cell line U93748 h, fluorescein quantitative PCR, Western blotting method to detect HOXA5 and protein The recombinant plasmid vector,.2., was screened out for the best effect of silent HOXA5, and the screened HOXA5 shRNA recombinant plasmid was transfected into U937 cells. CCK-8 and flow cytometry were used to detect the proliferation ability and cell cycle of U937 cells, cell cycle, apoptosis and Western blotting detection of apoptosis related protein Surviv. The expression changes of in and Caspase-3. Results 1. successfully constructed 3 pGPU6/GFP/Neo-HOXA5 recombinant plasmid carriers. After transfection of 48 fluorescent quantitative RT-PCR and Western blotting analysis, it was found that all 3 recombinant plasmid vectors could inhibit the expression of HOXA5, and shRNA3's silencing efficiency was the highest, and the recombinant plasmid vector was screened to transfect U937 cells, West The results of ern blotting and fluorescence quantitative RT-PCR assay showed that compared to the blank control group and the negative control group, the U937 cells transfected with the recombinant plasmid vector of pGPU6/GFP/Neo-HOXA5 shRNA-3 could obviously inhibit the expression of HOXA5 gene, and the silence efficiency could reach 70% (P0.05).CCK-8 method and found that U937 cells were transfected into 0,24,48,72 h respectively. HRNA could obviously inhibit the proliferation of U937 cells, and the growth inhibition rate increased gradually with the prolongation of transfection time (P0.05). Flow cytometry showed that the number of G0/G1 cells in the experimental group was more than that of the blank control group and the negative control group. The number of S phase cells decreased by.Annexin V-FITC double staining, and the blank control group, negative control group, HOXA5. The apoptosis rate of shRNA group was (7.36 + 1.63)%, (8.10 + 1.39)% and (26.52 + 2.35)%. The apoptosis rate of HOXA5 shRNA group was significantly higher than that of the other two groups (P0.05).Western blotting detection: HOXA5 shRNA interfered U937 cell 48 h, and the expression of HOXA5 shRNA group was significantly lower than that of negative control group and blank control group, and the expression of HOXA5 shRNA group was significantly lower than that of the negative control group and the blank control group. The expression of aspase-3 protein was significantly higher than that in the other two groups (P0.05). Conclusion: 1. the recombinant plasmid expressing vector pGPU6/GFP/Neo-HOXA5 shRNA-1, -2, -3 is successfully constructed, and the recombinant plasmid carrier,.2. recombinant expression vector pGPU6/ GFP/Neo-HOXA5-3, can be effectively silenced after transfection of U937 cells. The 5 gene inhibits the expression of HOXA5 mRNA and protein: RNA interference with the expression of HOXA5 gene can significantly inhibit the proliferation of U937 cells, induce cycle arrest and promote apoptosis. The latter may be related to the decrease of the expression of Survivin protein and the increase of the expression of Caspase-3 protein in the inhibition of the apoptosis gene Survivin.

【學(xué)位授予單位】：濱州醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R3416

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本文編號(hào)：1792186