人激肽释放酶重组腺相关病毒对人脐静脉内皮细胞的影响  

Effect of recombinant adeno-associated virus vetor carring human tissue kallikrein genet on human umbilical vein endothelial cells

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作  者:李世峰[1] 陈慧[1] 

机构地区:[1]福建省立医院,福建福州350001

出  处:《心血管康复医学杂志》2010年第6期578-582,F0004,共6页Chinese Journal of Cardiovascular Rehabilitation Medicine

基  金:福建省青年科技人才创新专项基金(2001J061)

摘  要:目的:构建携带人激肽释放酶(kallikrein,KK)基因的重组腺相关病毒载体,并导入体外培养的人脐静脉内皮细胞(HUVEC),观察其对缓激肽(BK)、一氧化氮(NO)、内皮素-1(ET-1)水平和内皮型一氧化氮合酶(eNOS)基因表达的影响。方法:(1)将KK基因定向克隆入腺相关病毒载体质粒pAAV-MCS中,构建成pAAV-KK;(2)分别将pAAV-KK及pAAV-LacZ与另外两种质粒pHelper、pAAV-RC通过脂质体共同转染AAV-293细胞,包装出rAAV-KK和rAAV-LacZ重组腺相关病毒。将rAAV-LacZ原液稀释成不同浓度梯度并感染HT-1080细胞,通过β-半乳糖苷酶染色测定病毒感染滴度,并观察LacZ传代HT-1080细胞的表达情况;(3)将rAAV-KK感染HUVEC,检测其对缓激肽、NO、ET-1水平和eNOS基因表达的影响。结果:(1)成功构建了带有KK目的基因的重组腺相关病毒载体和带有LacZ报告基因的重组腺相关病毒载体(对照载体);(2)rAAV-LacZ重组病毒以感染滴度6.2×107I U/ml感染HT-1080细胞后,LacZ基因能够在连续传代的HT-1080细胞内稳定持续表达;(3)与空白组、LacZ组(对照组)相比,KK感染组的HUVEC细胞培养液中BK[(2.864±1.36)pmol/ml∶(2.782±1.48)pmol/ml∶(6.576±2.08)pmol/ml]、NO[(16.42±1.02)μmol/L∶(16.46±1.25)μmol/L∶(21.28±1.46)μmol/L]水平明显增加,eNOS基因[(0.353±0.031)∶(0.364±0.049)∶(0.423±0.038)]表达明显增强,ET-1[(262.91±17.85)pg/ml∶(263.56±15.87)pg/ml∶(199.48±16.99)pg/ml]水平显著下降(P均<0.01)。结论:(1)成功构建了携带有人激肽释放酶基因的重组腺相关病毒,rAAV-KK感染体外培养的人脐静脉内皮细胞;(2)可使细胞分泌BK、NO,以及表达eNOS基因增加,ET-1减少,改善内皮细胞功能。Objective:(1) To construct recombinant adeno-associated virus(rAAV) vector carring the human kallikrein(KK) gene.and detect its titer;(2)The KK gene was transfered into HUVEC in vitro.The effect of KK gene on the level of bradykinin(BK) nitric oxide(NO) and endothelin-1(ET-1) and expressions of endothelial nitricoxide synthase(eNOS) gene were observed.Methods:(1)The KK gene was directional cloned into pAAV-MCS plasmid,constructed pAAV-KK;(2)Three plasmids(pAAV-KK or pAAV-LacZ,and pAAV-RC,pHelper) were co-transfect the AAV-293 cell with by lipofectamine 2000 and packed rAAV vector carring the KK gene or LacZ gene.The HT-1080 cell was infected by 6.2 ×107IU/ ml of rAAV-LacZ or rAAV-KK,then was stained by β-Galactosidase staining to estimate the titer of virus;(3)The cultured HUVEC in vitro was infected by rAAV-KK,and its effects on levels of BK,NO,ET-1 and expressions of eNOS gene were observed.Results:(1)The rAAV-KK and rAAV-LacZ vector were successfully construsted;(2)The LacZ gene could be stably expressed in the passaged HT-1080 cell;(3)Compared with blank group and rAAV-LacZ(control)group,the level of BK[(2.864±1.36)pmol/ml vs.(2.782±1.48) pmol/ml vs.(6.576±2.08) pmol/ml]、NO [(16.42±1.02) μmol/L vs.(16.46±1.25) μmol/L vs.(21.28±1.46) μmol/L] and the expression of eNOS gene [(0.353±0.031)vs.(0.364±0.049) vs.(0.423 0.038)] significantly increased while the level of ET-1[(262.91±17.85)pg/ml vs.(263.56±15.87) pg/ml vs.(199.48±16.99) pg/ml] significantly decreased in HUVEC cell of KK infected group,P〈0.01 all.Conclusions:(1)The rAAV-KK was successfully construscted and LacZ gene,as a foreign gene,could be stably expressed in the passaged HT-1080 cell;(2)The rAAV-KK could increase levels of BK,NO and expressions of eNOS gene and decrease level of ET-1,improve function of vascular endothelial cell in HUVEC cell infected from rAAV-KK.

关 键 词:激肽释放酶类 内皮细胞 腺病毒感染 

分 类 号:Q709[生物学—分子生物学]

 

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