Smad3 vector and keloid fibroblasts cultured Objective: To construct suppress keloid fibroblasts (KFB) Smad3 gene expression shRNA eukaryotic expressio" />
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The Expression of shRNA Suppressing Smad3 in KFB

Author: DanYang
Tutor: ShenWeiMin
School: Chongqing Medical University
Course: Surgery
Keywords: Liposome -mediated method Keloid fibroblasts (KFB) shRNA Smad3 Scar fibroblasts Smad3 Smad7 RNA interference shRNA Ⅰ collagen
CLC: R622
Type: Master's thesis
Year: 2009
Downloads: 42
Quote: 0
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Abstract


The first part of the shRNA-Smad3' dissertation">Smad3 vector and keloid fibroblasts cultured Objective: To construct suppress keloid fibroblasts (KFB) Smad3 gene expression shRNA eukaryotic expression vector and transfected into primary cultured scar fibroblasts get the best transfection conditions. Methods: preliminary screening test a pair of the most effective siRNA sequences recombination Smad3 shRNA expression plasmid. Liposome-mediated method, in different proportions, time and concentration, determined by fluorescence microscopy optimal transfection conditions. By tissue adherent primary method of keloid fibroblasts. Results: The sequencing gel electrophoresis analysis, successfully constructed shRNA-Smad3 vector, shRNA-Smad3 and liposome ratio was 1:1.8, in serum-free conditions, time 72h after transfection highest transfection efficiency. Primary keloid fibroblasts success. Conclusion: plasmid DNA liposomes added under proper ratio in a short time transfection, liposome transfection of primary cultures of human keloid fibroblasts can obtain a higher gene transfection rate, and the minimum cell death. Liposomes are of primary cultured human keloid fibroblasts ideal behavior transfection methods. Tissue explant can get keloid fibroblasts. The second part of the shRNA-Smad3 on keloid fibroblasts Smad3, COL1A2, and gene expression of Smad7 Objective: To study the shRNA-Smad3 right after KFB KFB transfected Smad3, Smad7 and COL1A2 expression. Methods: liposome-mediated method, the shRNA-Smad3 was transfected into scar fibroblasts by immunohistochemistry, RT-PCR and Western blotting was used to detect Smad3, Smad7 at different time points (0 ~ 9d) expression changed. Results: ① RT-PCR and Western blotting results confirm Smad3 shRNA vector was constructed successfully. ② After Smad3 shRNA transfection, with the time, KFB in Smad3 mRNA and protein expression were significantly reduced, to the strongest 72h. Optical density analysis compared with the control group there was significant difference (P lt; 0.05). Smad7 mRNA and protein expression were significantly higher (P lt; 0.05). Conclusion: After transfection of shRNA-Smad3, make KFB's Smad3mRNA and protein level was inhibited, also make Ⅰ collagen mRNA and protein expression decreased, Samd7 mRNA and protein was significantly increased, suggesting that Smad7 may play anti-Smad3 to the role of regulation.

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