摘要
目的以糖原合成酶激酶-3β（GSK-3β）特异的RNA干扰（RNAi）腺病毒表达载体作为研究工具，从转录后水平抑制GSK-3β基因表达，探讨Wnt/β-连环蛋白（Wnt/β-catenin）信号通路对人甲状腺细胞增殖的影响。方法利用体外同源重组技术构建短发夹RNA（shRNA）腺病毒表达载体，在HEK293A细胞中包装并扩增病毒、空斑实验法测定病毒滴度；RNAi腺病毒感染原代培养人甲状腺细胞，Western印迹法检测其对GSK-3β蛋白表达的抑制效应；利用构建的RNAi腺病毒抑制GSK-3β表达，BrdU法检测原代培养人甲状腺细胞增殖活性。结果成功构建了GSK-3β特异的RNAi腺病毒表达载体，获得高滴度的腺病毒液；RNAi腺病毒感染原代培养人甲状腺细胞后，GSK-3β蛋白的表达受到明显抑制，β-catenin蛋白表达显著增加（1．324±0．057vs0．935±0．029，P〈0．05）；腺病毒感染原代培养人甲状腺细胞1、3、5、7d后，RNAi腺病毒组比空病毒组细胞增殖率明显增加（1．473±0．035vs1．312±0．043、1．226±0．045vs0．959±0．066、0．776±0．041vs0．605±0．054、0．337±0．018vs0．259±0．021，P〈0．05）。结论构建的目的RNAi腺病毒表达载体能有效抑制GSK-3β基因的表达；Wnt／β-catenin信号通路在人甲状腺细胞的增殖中有重要影响。
Objective To construct RNAi recombinant adenoviral expressive vectors specific to glycogen synthase kinase-3β （ GSK-3β） and to observe its gene knockdown effect on the expression of GSK- 3β, and to explore the effect of Wnt/β-eatenin pathway on the proliferation of human thyrocytes using the RNAi adenovirus vector. Methods An adenovirus plasmid that contained the RNAi cassette targeting the GSK-3β gene was constructed by homologous recombination and cloning techniques, transfected into human embryo kidney （HEK） 293A ceils to product adenovirus, and then was used to infect the HEK293A cells to amplify the adenoviral stock. Plaque forming assay was used to titer the adenoviral stock . Normal human thyrocytes fart from thyroid adenoma were obtained during resection of adenoma, cultured, and infected by the GSK-3β specific RNAi adenovirus. The GSK-3β gene silencing effect induced by the RNAi adenovirus was detected by Western blotting 0, 24, 48, 72, 120, and 144 hours later. BrdU method was used to detect the cell proliferation. Another HEK293A cells were divided into 3 groups： infected with recombinant adenovirus plasmid Ad-1457, infected with un-recombinant framework plasmid pAd-DEST, and un-infected. 72 hours later Western blotting was used to examine the level of β-catenin. Results The GSK-3β expression of the thyrocytes infected with the recombinant adenovirus plasmid Ad-1457 were significantly lower than those of the thyrocytes infected with Ad-DEST （ all P 〈 0.05 ）. The expression of β-catenin of the thyrocytes infected with Ad-DEST was significantly higher than those of the Ad-DEST group and un-infected group （ both P 〈 0.05 ）. BrdU assay suggested that the proliferation rates 1,3, 5, and 7 days after infection of the thyrocytes infected with Ad1457 plasmid were significantly higher than those of the thyrocytes infected with the plasmid pAd-DEST （ all P 〈 0.05 ）. Conclusion RNAi adenovirus is an important tool inhibiting the expression of target gene efficiently. The Wnt/β-catenin pathway plays an important role in the regulation of proliferation of human thyrocytes.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2008年第40期2821-2825,共5页
National Medical Journal of China
关键词
信号传递
人甲状腺细胞
增殖
Signal transduction
Human thyrocytes
Proliferation
