Smad 6和Smad 7基因腺相关病毒载体的构建及其表达

目的 :构建Smad 6和Smad 7基因腺相关病毒 (AAV)载体 ,并转染到人肾小管上皮细胞中表达。方法 :利用基因重组技术 ,采用BamHI和XhoI双酶分别将pcDNA3中的目的基因Smad6 /flag和Smad 7/flag融合基因片断切出 ,再克隆到质粒pAAV MCS中 ,构建重组质粒pAA Smad 6 /flag和pAA Smad 7/flag。采用磷酸钙沉淀法 ,以重组质粒或pAAV LacZ以及pAAV RC、pHelper共转染HEK2 93细胞 ,产生均有传染性的病毒颗粒。再将此病毒颗粒转染到培养的人肾小管细胞中 ,用免疫组化法检测其Smad 6和Smad 7转移基因的表达。结果 :成功地构建Smad 6和Smad 7基因的腺相关病毒载体 ,并可在肾小管上皮细胞中表达Smad 6和Smad 7。结论 :肾小管细胞能稳定、高效表达外源Smad 6和Smad 7,为今后建立Smad 6和Smad 7AAV基因治疗体内肾纤维化的模型奠定了良好的基础

Construction and expression of adeno-associated virus vectors of Smad 6 and Smad 7 genes in human renal tubule epithelial cells

AIM: 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. METHODS: The plasmids pcDNA3 Smad 6/flag and pcDNA3 Smad 7/flag were digested with Bam H 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. RESULTS: The recombinant AAV vectors of Smad 6 or Smad 7 genes were constructed and expressed in the HKCs successfully. CONCLUSION: 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.