【摘要】：目的:本課題旨在構(gòu)建針對HOXA5sh RNA真核表達載體p RNAT-GFP-Neo-HOXA5沉默HOXA5基因表達,探討其對Jurkat細胞Livin蛋白、Smac蛋白表達的影響,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)計合成三條針對HOXA5基因不同位點的si RNA序列,并篩選有效干擾HOXA5基因表達的特異性si RNA序列,構(gòu)建靶向HOXA5的sh RNA真核表達載體p RNAT-GFP-Neo-si HOXA5。2、實驗分組:取對數(shù)期細胞(約3x107/ml),將Jurkat細胞分為三組:實驗組(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(僅加等量細胞及培養(yǎng)基)。3、轉(zhuǎn)染后FQ-PCR:通過脂質(zhì)體LipofectamineTM2000介導si RNA轉(zhuǎn)染Jurkat細胞,48h后通過FQ-PCR和Western blot測定實驗組、陰性對照組和空白對照組三組Jurkat細胞HOXA5m RNA和蛋白以及Livin、Smac蛋白相對表達水平。結(jié)果:1、設(shè)計并構(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基因的表達量在實驗組明顯下調(diào),轉(zhuǎn)染后實驗組各組Jurkat細胞基因表達受抑制,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,實驗組(p RNAT-GFP-Neo-HOXA5C)HOXA5m RNA表達水平(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,實驗組(p RNAT-GFP-Neo-HOXA5C)Jurkat細胞HOXA5蛋白表達水平(0.18±0.01)較明顯下降(P0.05)。3、實驗組livin蛋白表達量(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、實驗組Smac蛋白表達量(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表達載體,能夠有效沉默HOXA5基因的表達,下調(diào)HOXA5表達能直接誘導Livin蛋白表達下調(diào)和Smac蛋白表達上調(diào),并促進細胞凋亡;2、本研究為急性淋巴細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.
【學位授予單位】：西南醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R733.7

【參考文獻】

相關(guān)期刊論文 前10條

1 羅靜;;RNA干擾在抗腫瘤中的作用研究進展[J];新鄉(xiāng)醫(yī)學院學報;2017年04期

2 姚明;王彥;徐暢;劉強;;Smac與腫瘤的放化療增敏[J];生命科學;2016年11期

3 陳文慶;張依軍;蔡三立;寇國鋒;劉剛;;凋亡蛋白Smac與食管癌[J];中國實驗診斷學;2016年11期

4 裴蕾;王志強;賀璇;周英瓊;;同源異型盒基因HOXA5在腫瘤及其相關(guān)疾病發(fā)生發(fā)展中的作用[J];中外女性健康研究;2016年21期

5 熊凱;薛麗香;;CBX4與HOXA5在慢性粒細胞白血病中的表達及相互作用研究[J];中國生物化學與分子生物學報;2016年09期

6 馬大驊;黃楷;姜夢迪;徐菲菲;管藝貝;程楓;;凋亡抑制蛋白Livin在腫瘤中的作用及其診斷和治療應(yīng)用進展[J];上海交通大學學報(醫(yī)學版);2016年05期

7 黃瑩瑩;劉浩;;以IAPs為靶點的抗腫瘤研究進展[J];中國藥理學通報;2016年05期

8 祝平;沈樹娜;;子宮內(nèi)膜異位癥中Livin和Smac的表達及相關(guān)性研究[J];中國婦幼健康研究;2016年04期

9 楊立軍;鄭文武;周安傳;蘇曉哲;吳亞東;李s，

本文編號：2506553