Huang Yun-Jian, Zhao Jing-Hong, Yang Tang-Jun, Zhang Jin-Hai, Cai Wen-Qin
Department of Nephrology, Xinqiao Hospital, Chongqing 400037, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2004 May;20(3):274-7.
To construct the adeno-associated virus(AAV) vectors of Smad 6 and Smad 7 genes and observe their expressions in human renal tubule epithelial cells.
The plasmids pcDNA3-Smad 6/flag and pcDNA3-Smad 7/flag were digested with BamH I and Xho I. Then the Smad 6/flag and Smad 7/flag gene fragments were cloned into plasmid pAAV-MCS, respectively to construct the recombinant pAAV-Smad 6/flag and pAAV-Smad 7/flag plasmids. The recombinant expression plasmid or pAAV-LacZ plasmid were co-transfected into the HEK 293 cells with pHelper and pAAV-RC by calcium-phosphate precipitation method. Recombinant AAV-2 viral particles were prepared from infected HEK293 cells and then were used to infect human renal tubule epithelial cells (HKCs), The expressions of Smad 6 and Smad 7 in HKCs were demonstrated by immunocytochemical staining.
The recombinant AAV vectors of Smad 6 or Smad 7 genes were constructed and expressed in the HKCs successfully.
These results indicate that AAV can deliver Smad 6 and Smad 7 genes to renal cells in-vitro, suggesting the recombinant AAV can be used for gene therapy of renal fibrosis.
构建Smad 6和Smad 7基因的腺相关病毒(AAV)载体,并观察它们在人肾小管上皮细胞中的表达。
用BamH I和Xho I酶切质粒pcDNA3-Smad 6/flag和pcDNA3-Smad 7/flag。然后将Smad 6/flag和Smad 7/flag基因片段分别克隆到质粒pAAV-MCS中,构建重组pAAV-Smad 6/flag和pAAV-Smad 7/flag质粒。通过磷酸钙沉淀法将重组表达质粒或pAAV-LacZ质粒与pHelper和pAAV-RC共转染到人胚肾293(HEK 293)细胞中。从感染的HEK293细胞中制备重组AAV-2病毒颗粒,然后用于感染人肾小管上皮细胞(HKCs),通过免疫细胞化学染色检测HKCs中Smad 6和Smad 7的表达。
成功构建了Smad 6或Smad 7基因的重组AAV载体,并在HKCs中表达。
这些结果表明,AAV可以在体外将Smad 6和Smad 7基因递送至肾细胞,提示重组AAV可用于肾纤维化的基因治疗。