靶向沉默转化生长因子β1表达的短发夹RNA的筛选

Screening of short hairpin RNA targeting TGF-β1

目的：筛选可靶向沉默转化生长因子β1（TGF-β1）表达的短发夹RNA（shRNA）。方法：设计和制备靶向沉默TGF-β1的5条shRNA,并构建靶向沉默TGF-β1的p-Genesil-shRNA真核表达重组质粒及含无关shRNA的pGenesil-1-shRNA-vect对照质粒,酶切和测序方法进行鉴定。通过脂质体介导分别将p-Genesil-1-shRNA-vect质粒和各重组表达质粒转染入高糖或AngⅡ环境激活的HKC细胞（分别命名为p-Genesil-1-vect细胞及p-Genesil-shRNA1-5细胞）,Western blot方法检测沉默TGF-β1表达的效果。结果：酶切和测序结果显示,5种重组质粒均可切出与预计相符的目的片段,所有shRNA编码序列与设计一致。与HKC细胞相比,高糖或AngⅡ刺激的HKC细胞和pGenesil-1-vect细胞中TGF-β1表达均增高（F=74.188,P〈0.001）,而后二者之间TGF-β1表达差异无统计学意义（P〉0.05）。与高糖或AngⅡ刺激的p-Genesil-1-vect细胞相比,高糖或AngⅡ刺激的各组p-Genesil-shRNA细胞中TGF-β1表达均降低（F=139.695,P〈0.001）。结论：筛选出1条可靶向沉默TGF-β1表达的shRNA。

Aim： To screen the short hairpin RNA（ shRNA） targeting TGF-β1.Methods： Covering the c DNA fulllength of TGF-β1 gene,5 pairs of shRNA targeting TGF-β1 mRNA were designed and obtained.The eukaryotic expression recombinant plasmid（ p-Genesil-shRNA 1,2,3,4 and 5） targeting TGF-β1 and unrelated shRNA（ p-Genesil-1-shRNAvect） were constructed.The plasmids were identified by enzyme digestion and gene sequencing respectively.The above mentioned eukaryotic expression recombinant plasmids were transfected into HKC cells which had been activated by high glucose or AngⅡ via liposomes,then named p-Genesil-1-vect cells,p-Genesil-shRNA 1,2,3,4 and 5 cells.The shRNA targeting TGF-β1 was screened through detecting the changes of expression of TGF-β1 by Western blot.Results： The results of enzyme digestion showed that the length of digestion fragments was consistent with expected length respectively.The results of gene sequencing showed that,compared with designed sequences,gene coding sequences of shRNA were exactly consistent.The results of Western blot showed that： compared with HKC cells,the expression of TGF-β1 in HKC cells stimulated by high glucose or AngⅡ and p-Genesil-1-vect cells was significantly increased（ F = 74.188,P〈 0.001）.However,the expression of TGF-β1 in HKC cells stimulated by high glucose or AngⅡ and p-Genesil-1-vect cells had no significant difference（ P〈 0.05）.Compared with p-Genesil-1-vect cells stimulated by high glucose or AngⅡ,the expression of TGF-β1 in p-Genesil-shRNA cells were significantly depressed（ F = 139.695,P〈 0.001）.Conclusion： One pair of shRNA which could efficiently silence TGF-β1 expression has been screened.