本文選題：核糖核酸酶抑制因子 + 人臍靜脈內(nèi)皮細(xì)胞��； 參考：《重慶醫(yī)科大學(xué)》2012年碩士論文

【摘要】：目的構(gòu)建融合表達(dá)載體pcDNA3.1 RI，并檢測核糖核酸酶抑制因子(ribonuclease inhibitor，RI)基因與血管生成素(angiogenin, ANG)的關(guān)系及對人臍靜脈內(nèi)皮細(xì)胞(human umbilical vein endothelialcells, HUVEC)增殖及遷移能的影響。 方法用RT-PCR方法擴(kuò)增了RI基因，酶切后將其插入pcDNA3.1 ，構(gòu)建融合表達(dá)載體pcDNA3.1 RI，在脂質(zhì)體介導(dǎo)下轉(zhuǎn)染人臍靜脈內(nèi)皮細(xì)胞HUVEC，，通過RT-PCR檢測RI、ANG基因的mRNA表達(dá)水平，Western blot檢測RI、ANG及影響內(nèi)皮細(xì)胞遷移能力重要的2個蛋白MMP-2、MMP-9的表達(dá)水平，；CO-IP法檢測ANG和RI的相互作用，MTT法檢測細(xì)胞的增殖活力，流式細(xì)胞術(shù)檢測細(xì)胞周期的分布。 結(jié)果真核表達(dá)質(zhì)粒構(gòu)建成功；實驗組pcDNA3.1-RI細(xì)胞RI基因的mRNA及蛋白的表達(dá)較2個對照組(轉(zhuǎn)pcDNA3.1 空載體組和未轉(zhuǎn)染質(zhì)粒組)比較,均呈顯著性增加(P0.05)，轉(zhuǎn)染RI基因的細(xì)胞ANG基因的mRNA及蛋白的表達(dá)均降低(P0.05)；MMP-2、MMP-9基因的蛋白表達(dá)水平降低(P0.05)；CO-IP法檢測到ANG和RI在細(xì)胞內(nèi)能結(jié)合；轉(zhuǎn)染pcDNA3.1 RI質(zhì)粒到HUVEC細(xì)胞后細(xì)胞的增殖活力明顯降低(P0.05),G0～G1期比例明顯增加，S期減少。 結(jié)論成功構(gòu)建的真核表達(dá)質(zhì)粒能顯著增加RI基因及其蛋白水平的表達(dá)，RI可以直接在轉(zhuǎn)錄水平上降低ANG的表達(dá)，在細(xì)胞內(nèi)與ANG結(jié)合，從而影響內(nèi)皮細(xì)胞的增殖、遷移能力。
[Abstract]:Objective to construct the fusion expression vector pcDNA3.1 ri and to detect the relationship between ribonuclease inhibitor ribonuclease receptor and angiogenin (Ang) and its effect on proliferation and migration of human umbilical vein endothelial cells (HUVECs). Methods RI gene was amplified by RT-PCR. The fusion expression vector pcDNA3.1 was constructed and transfected into human umbilical vein endothelial cells (HUVECs) mediated by liposome. The level of mRNA expression of RIANG gene was detected by RT-PCR. It is important to detect RIANG by Western blot and to affect the migration ability of HUVECs. The expression level of MMP-2MMP 9 was detected by CO-IP method. The interaction between ANG and RI was detected by MTT assay and the proliferative activity of cells was detected by MTT assay. The distribution of cell cycle was detected by flow cytometry. Results the expression of mRNA and protein of RI gene in pcDNA3.1-RI cells in the experimental group was compared with that in the control group (transfer pcDNA3.1 empty vector group and untransfected plasmid group), and the expression of RI gene in the experimental group was compared with that in the control group. The expression of mRNA and protein of ANG gene in the cells transfected with RI gene decreased, and the protein expression level of MMP-2MMP-9 gene decreased. The binding of ANG and RI in cells was detected by P0.05 ANG CO-IP method. After transfection of pcDNA3.1 / RI plasmid into HUVEC cells, the proliferative activity of the cells decreased significantly. The ratio of G _ 0 / G _ (1) phase of P0.05G _ (0) / G _ (1) increased significantly. Conclusion the successfully constructed eukaryotic expression plasmid can significantly increase the expression of RI gene and its protein. RI can directly reduce the expression of ANG at the transcriptional level and bind to ANG in cells, thus affecting the proliferation and migration ability of endothelial cells.
【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】：2012
【分類號】：R346

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