摘要
目的构建糖原合成酶激酶3β(GSK-3β)特异的RNA干扰(RNAi)腺病毒,观察其抑制高游离脂肪酸(FFA)诱导下人脐静脉内皮细胞(HUVEC)GSK-3β的表达情况。方法利用体外同源重组技术构建GSK-3β特异的siRNA腺病毒表达载体,在HEK293A细胞中包装并扩增病毒、空斑以实验法测定病毒滴度。GSK-3β特异的siRNA腺病毒感染HUVEC,以Western印记法测定其对GSK-3β蛋白表达的影响。结果成功构建了GSK-3β特异的siRNA腺病毒,获得高滴度的腺病毒液;构建的重组腺病毒感染HUVEC,可显著抑制正常培养及游离脂肪酸诱导下的细胞的GSK-3β蛋白的表达,Ad-DEST组与Ad-640组比较,Ad-640组明显下降(P<0.05)。结论构建的GSK3β特异的RNAi腺病毒能有效抑制HUVECGSK-3β蛋白的表达,有可能保护高脂诱导的HUVEC损伤。
Objective To construct RNAi recombinant adenovirus expressive vectors specific to GSK-3β and explore ff it can inhibit the GSK-3β expression of human umbilical vein endothelial ceils induced by high free fatty acid. Methods Homologous recombination and cloning techniques were used to construct RNAi recombinant adenovirus expressive vectors specific to GSK-3β. Then, the adenovirus plasmids was transfected into HEK293A ceils to produce adenovirus and amplify the adenovirus stock. Plaque forming assay was used to titer the adenovirus stock. The GSK-3βexpression were detected by Western blot. Results The RNAi adenovirus vectors specific to GSK-3β were successfully produced with high titer. The expression of GSK-3β protein could be down-regulated efficiently by the RNAi adenovirus in HUVEC both in the condition of normol and high free fatty acid （ P 〈 0. 05 ）. Conclusion RNAi adenovirus is an important tool that inhibit the expression of GSK-3β efficiently. It may partly protect HUVEC from impairment which induced by FFAs.
出处
《中国临床新医学》
2010年第1期1-3,共3页
CHINESE JOURNAL OF NEW CLINICAL MEDICINE
基金
福建省自然科学基金(2007J0072)
关键词
RNA干扰
糖原合成酶激酶3Β
游离脂肪酸
人脐静脉内皮细胞
RNA interference
Glycogen synthetase kinase-3 beta （GSK-3β）
Free fatty acid（FFA）
Human umbilical vein endothelial cells（HUVEC）
