本文選題：血紅素加氧酶- + 慢病毒��； 參考：《中國(guó)醫(yī)學(xué)科學(xué)院學(xué)報(bào)》2014年04期

【摘要】：目的構(gòu)建穩(wěn)定表達(dá)人血紅素加氧酶-1(HO-1)的細(xì)胞系,探討內(nèi)源性過(guò)表達(dá)HO-1對(duì)抗細(xì)胞氧化損傷的作用。方法 PCR擴(kuò)增HO-1基因并將其克隆至改造的慢病毒載體pLentiLox3.7中,構(gòu)建攜帶目的基因的重組質(zhì)粒。用重組質(zhì)粒轉(zhuǎn)染HEK293T細(xì)胞,并用Western blot檢測(cè)HO-1的表達(dá)。將重組質(zhì)粒與慢病毒輔助質(zhì)粒(plp1、plp2、VSVG)共同轉(zhuǎn)染HEK293T細(xì)胞,包裝有活力的慢病毒。用攜帶HO-1的慢病毒感染HEK293T細(xì)胞,篩選出能穩(wěn)定表達(dá)目的基因的單克隆,并行Western blot檢測(cè)HO-1的表達(dá)。將過(guò)氧化氫加入正常的及過(guò)表達(dá)HO-1的HEK293T細(xì)胞和人臍靜脈內(nèi)皮細(xì)胞,檢測(cè)細(xì)胞內(nèi)活性氧的含量。結(jié)果成功篩選出能穩(wěn)定表達(dá)HO-1的單克隆細(xì)胞系并擴(kuò)大培養(yǎng),Western blot檢測(cè)其HO-1的表達(dá)明顯升高。加入過(guò)氧化氫后,過(guò)表達(dá)HO-1的HEK293T細(xì)胞和人臍靜脈內(nèi)皮細(xì)胞內(nèi)的活性氧明顯減少。結(jié)論成功構(gòu)建了能穩(wěn)定表達(dá)人HO-1的細(xì)胞系,內(nèi)源性過(guò)表達(dá)HO-1能夠?qū)辜?xì)胞的氧化損傷。
[Abstract]:Aim to construct a cell line stably expressing human heme oxygenase-1 (HO-1) and to investigate the role of endogenous overexpression of HO-1 in the prevention of oxidative damage. Methods the HO-1 gene was amplified by PCR and cloned into the modified lentivirus vector pLentiLox3.7 to construct the recombinant plasmid carrying the target gene. HEK293T cells were transfected with recombinant plasmid and the expression of HO-1 was detected by Western blot. The recombinant plasmid was co-transfected with lentivirus-assisted plasmids (plp1 / plp2hVSVG) into HEK293T cells and the active lentivirus was packaged. After infection of HEK293T cells with lentivirus carrying HO-1, monoclonal clones were screened for stable expression of the target gene, and HO-1 expression was detected by Western blot. Hydrogen peroxide was added to normal and overexpressed HEK293T cells and human umbilical vein endothelial cells to detect the content of reactive oxygen species in the cells. Results Monoclonal cell lines with stable expression of HO-1 were successfully screened and their HO-1 expression was detected by Western blot. After adding hydrogen peroxide, reactive oxygen species (Ros) in HEK293T cells and human umbilical vein endothelial cells (HUVEC) overexpression of HO-1 decreased significantly. Conclusion A cell line with stable expression of human HO-1 was successfully constructed, and endogenous overexpression of HO-1 could antagonize oxidative damage.
【作者單位】： 中國(guó)醫(yī)學(xué)科學(xué)院北京協(xié)和醫(yī)學(xué)院北京協(xié)和醫(yī)院血管外科;中國(guó)醫(yī)學(xué)科學(xué)院北京協(xié)和醫(yī)學(xué)院基礎(chǔ)醫(yī)學(xué)研究所醫(yī)學(xué)分子生物學(xué)國(guó)家重點(diǎn)實(shí)驗(yàn)室;
【基金】：北京市自然科學(xué)基金(7122145)~~
【分類號(hào)】：R346

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