【摘要】：目的:本課題旨在構(gòu)建針對HOXA5sh RNA真核表達(dá)載體p RNAT-GFP-Neo-HOXA5沉默HOXA5基因表達(dá),探討其對Jurkat細(xì)胞Livin蛋白、Smac蛋白表達(dá)的影響,為白血病的靶向治療提供理論依據(jù)和線索。方法:1、根據(jù)HOXA5基因的m RNA序列,設(shè)計(jì)合成三條針對HOXA5基因不同位點(diǎn)的si RNA序列,并篩選有效干擾HOXA5基因表達(dá)的特異性si RNA序列,構(gòu)建靶向HOXA5的sh RNA真核表達(dá)載體p RNAT-GFP-Neo-si HOXA5。2、實(shí)驗(yàn)分組:取對數(shù)期細(xì)胞(約3x107/ml),將Jurkat細(xì)胞分為三組:實(shí)驗(yàn)組(脂質(zhì)體轉(zhuǎn)染靶向HOXA5的si RNA)、陰性對照組(脂質(zhì)體轉(zhuǎn)染陰性對照si RNA)和空白對照組(僅加等量細(xì)胞及培養(yǎng)基)。3、轉(zhuǎn)染后FQ-PCR:通過脂質(zhì)體LipofectamineTM2000介導(dǎo)si RNA轉(zhuǎn)染Jurkat細(xì)胞,48h后通過FQ-PCR和Western blot測定實(shí)驗(yàn)組、陰性對照組和空白對照組三組Jurkat細(xì)胞HOXA5m RNA和蛋白以及Livin、Smac蛋白相對表達(dá)水平。結(jié)果:1、設(shè)計(jì)并構(gòu)建針對HOXA5基因的短發(fā)卡RNA(Short hairpin,sh RNA),與空白對照組和陰性對照組作比較,HOXA5基因的表達(dá)量在實(shí)驗(yàn)組明顯下調(diào),轉(zhuǎn)染后實(shí)驗(yàn)組各組Jurkat細(xì)胞基因表達(dá)受抑制,抑制率分別為:p R N A T-G F P-N e o-H O X A 5 A(2 4.6 2±2.3 4)%,p RNAT-GFP-Neo-HOXA5B(35.07±3.21)%,p RNAT-GFP-Neo-HOXA5C(70.89±6.41)%,其中序列p RNAT-GFP-Neo-HOXA5C的效果最為顯著。2、成功構(gòu)建了有效沉默HOXA5基因的質(zhì)粒表達(dá)載體;與陰性對照組(1.34±0.16)%和空白對照組(1.29±0.21)%相比,實(shí)驗(yàn)組(p RNAT-GFP-Neo-HOXA5C)HOXA5m RNA表達(dá)水平(0.39±0.01)%明顯下降(P0.05)。與陰性對照組(0.84±0.02)及空白對照組(0.85±0.01)相比,實(shí)驗(yàn)組(p RNAT-GFP-Neo-HOXA5C)Jurkat細(xì)胞HOXA5蛋白表達(dá)水平(0.18±0.01)較明顯下降(P0.05)。3、實(shí)驗(yàn)組livin蛋白表達(dá)量(0.20±0.02)明顯低于陰性對照組(1.45±0.01)和空白對照組(1.33±0.01)(P0.05),而陰性對照組和空白對照組比較無明顯差異(PO.05);4、實(shí)驗(yàn)組Smac蛋白表達(dá)量(1.26±0.03)明顯高于陰性對照組(0.87±0.03)和空白對照組(0.86±0.03)(P0.05),而陰性對照組和空白對照組比較無明顯差異(PO.05)。結(jié)論:1、成功構(gòu)建的針對HOXA5基因的si RNA表達(dá)載體,能夠有效沉默HOXA5基因的表達(dá),下調(diào)HOXA5表達(dá)能直接誘導(dǎo)Livin蛋白表達(dá)下調(diào)和Smac蛋白表達(dá)上調(diào),并促進(jìn)細(xì)胞凋亡;2、本研究為急性淋巴細(xì)胞白血病靶向治療提供了新的理論依據(jù)。
[Abstract]:Objective: to construct HOXA5sh RNA eukaryotic expression vector p RNAT-GFP-Neo-HOXA5 to silence HOXA5 gene expression, to explore its effect on the expression of Livin protein and Smac protein in Jurkat cells, and to provide theoretical basis and clue for targeted therapy of leukemia. Methods: 1. According to the m RNA sequence of HOXA5 gene, three si RNA sequences targeting different sites of HOXA5 gene were designed and synthesized, and the specific si RNA sequences which effectively interfered with the expression of HOXA5 gene were screened. The sh RNA eukaryotic expression vector pRNAT-GFP-Neo-si HOXA5.2, targeting HOXA5 was constructed. Logarithmic cells (about 3x107/ml) were selected and divided into three groups: experimental group (si RNA), targeting HOXA5 by liposomes). Negative control group (negative control group si RNA) and blank control group (only add the same amount of cells and culture medium). 3. The relative expression of HOXA5m RNA, protein and Livin,Smac protein in Jurkat cells was measured by FQ-PCR and Western blot 48 hours later, and the relative expression of HOXA5m RNA, protein and Livin,Smac protein in Jurkat cells was measured by FQ-PCR and Western blot 48 hours later. Results: 1. Compared with the blank control group and the negative control group, the expression of HOXA5 gene was significantly down-regulated in the experimental group. After transfection, the gene expression of Jurkat cells in the experimental group was inhibited, and the inhibition rate was (24.62 鹵2.34)%, respectively. P RNAT-GFP-Neo-HOXA5B (35.07 鹵3.21)%, p RNAT-GFP-Neo-HOXA5C (70.89 鹵6.41)%, among which the effect of sequence p RNAT-GFP-Neo-HOXA5C was the most significant. 2. The plasmid expression vector which effectively silenced HOXA5 gene was successfully constructed. Compared with the negative control group (1.34 鹵0.16)% and the blank control group (1.29 鹵0.21)%, the expression level of HOXA5m RNA in the experimental group (p RNAT-GFP-Neo-HOXA5C) was significantly lower than that in the negative control group (1.34 鹵0.16)% and the blank control group (1.29 鹵0.21)% (P 0.05). Compared with the negative control group (0.84 鹵0.002) and the blank control group (0.85 鹵0.001), the expression of HOXA5 protein in Jurkat cells in the experimental group (p RNAT-GFP-Neo-HOXA5C) was significantly lower than that in the negative control group (0.84 鹵0.002) and the blank control group (0.85 鹵0.001). 3. The expression of livin protein in the experimental group was significantly lower than that in the negative control group (1.45 鹵0.001) and the blank control group (1.33 鹵0.001). There was no significant difference between the negative control group and the blank control group (PO.05). 4. The expression of Smac protein in the experimental group (1.26 鹵0.03) was significantly higher than that in the negative control group (0.87 鹵0.03) and the blank control group (0.86 鹵0.03) (P 0.05), but there was no significant difference between the negative control group and the blank control group (PO.05). Conclusion: 1. The successfully constructed si RNA expression vector targeting HOXA5 gene can effectively silence the expression of HOXA5 gene, down-regulate the expression of HOXA5 can directly induce the down-regulation of Livin protein expression and the up-regulation of Smac protein expression, and promote apoptosis. 2, this study provides a new theoretical basis for targeted therapy of acute lymphoblastic leukemia.
【學(xué)位授予單位】：西南醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R733.7

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