Altering equine corneal fibroblast differentiation through Smad gene transfer.
Altering equine corneal fibroblast differentiation through Smad gene transfer.
复制标题
DOI：
10.1111/vop.12485
复制
发表时间：
2018-03
影响因子：
1.6
通讯作者：
Mohan RR
中科院分区：
文献类型：
--
作者：
Marlo TL;Giuliano EA;Tripathi R;Sharma A;Mohan RR
探讨Smad基因单独或联合表达对马角膜成纤维细胞（ECF）向肌成纤维细胞（ECM）分化的影响。具体地，我们试图检查在（a）单独沉默Smad 2、3和4促纤维化基因和（B）过表达抗纤维化Smad 7基因以及与促纤维化和抗纤维化Smad基因组合后的ECF分化。如前所述产生ECF原代培养物。使用Lipofectamine 2000™或Lipofectamine BLOCK-iT™，用沉默Smad 2、3或4基因表达的单个质粒或与过表达Smad 7的质粒组合转染ECF。然后将Smad转染的克隆暴露于TGF-β1以诱导分化为肌成纤维细胞。免疫荧光和qRT-PCR技术通过测量α平滑肌肌动蛋白（ECM转分化的已知标志物）来定量ECF分化为ECM的水平。单个Smad 2、3或4基因的沉默或Smad 7的过表达显示ECF转分化的显著抑制（73-83%减少）。Smad 2的沉默在（a）中显示出对ECF转分化的最大抑制，因此用于组合基因转移测试。Smad 7过表达和Smad 2沉默的组合基因转移显著减弱了ECF的分化，但与单独Smad 7过表达相比，其水平并不显著。利用基因转移技术沉默促纤维化的Smad基因、过表达抗纤维化的Smad基因或二者联合应用是控制TGF-β1介导的马成纤维细胞纤维化的新策略。在本研究中，联合基因治疗并不比单一基因治疗更好。
To explore the impact of equine corneal fibroblast (ECF) to myofibroblast (ECM) differentiation by altering the expression of the Smad genes either individually or in combination. Specifically, we sought to examine the ECF differentiation after (a) silencing of Smad 2, 3 and 4 pro-fibrotic genes individually and (b) over-expression of anti-fibrotic Smad 7 gene and in a combination with pro- and anti-fibrotic Smad genes. ECF primary cultures were generated as previously described. ECFs were transfected with individual plasmids which silenced gene expression of either Smad2, 3, or 4 or in combination with a plasmid over-expressing Smad7 using Lipofectamine 2000™ or Lipofectamine BLOCK-iT™. Smad-transfected clones were then exposed to TGF-β1 to induce differentiation to myofibroblasts. Immunofluorescence and qRT-PCR techniques quantified levels of ECF differentiation to ECM by measuring alpha smooth muscle actin, a known marker of ECM transdifferentiation. Silencing of individual Smad2, 3, or 4 genes or overexpression of Smad7 showed significant inhibition of ECF transdifferentiation (73–83% reduction). Silencing of Smad2 showed the greatest inhibition of ECF transdifferentiation in (a) and was therefore utilized for the combination gene transfer testing. The combination gene transfer consisting of Smad7 overexpression and Smad2 silencing attenuated ECF differentiation significantly, however the level was not significant compared to the overexpression of Smad7 individually. Using gene transfer technology involving pro-fibrotic Smad silencing, anti-fibrotic Smad over-expression or its combination is a novel strategy to control TGF-β1 mediated fibrosis in equine fibroblasts. Combination gene therapy was not better than single gene therapy in this study.
登录
查看更多内容
影响因子：
4.3
作者：
Chen, Jingjing;Zeng, Tao;Zhao, Xiulan
通讯作者：
Zhao, Xiulan
影响因子：
1.6
作者：
Donnelly, Kevin S.;Giuliano, Elizabeth A.;Mohan, Rajiv R.
通讯作者：
Mohan, Rajiv R.
影响因子：
1.6
作者：
Donnelly, Kevin S.;Giuliano, Elizabeth A.;Mohan, Rajiv R.
通讯作者：
Mohan, Rajiv R.
影响因子：
3.4
作者：
Jester, JV;Huang, JY;Cavanagh, HD
通讯作者：
Cavanagh, HD
影响因子：
1.6
作者：
Clode, Alison;Davis, Jennifer;Gilger, Brian
通讯作者：
Gilger, Brian