[Changes of Smad2 and type I collagen expressions on cultured human Tenon's capsule fibroblasts transfected with Smad7 vector].
The purposes of this research is to investigate Smad2, phosphorylated Smad2 (p-Smad2) and type I collagen expressions in the cultured human Tenon's capsular fibroblasts (HTFs) transfected with Smad7 vector and to elucidate the mechanism of Smad7 in blocking tissue fibrosis after filtration surgery. Nucleofector was used to transfect Smad7 vector into HTFs. Reverse transcription real-time quantitive polymerase chain reaction (real-time RT-PCR) was then used to detect Smad7 mRNA expression levels. The expressions of HA-probe fusion protein, Smad2, p-Smad2, alpha2-type I procollagen (COL1A2) mRNA and carboxyterminal propeptide of type I procollagen (PICP) were determined by real-time RT-PCR, Western blot, cellular immunofluorescence assay and radioimmunoassay. The items mentioned above were also detected after HTFs being stimulated by TGF-beta2 (10 ng/ml). HTFs and vector-HTFs were used as control groups. Clones with Smad7 overexpression were successfully established. Results of real-time RT-PCR proved that there was no difference of Smad2 mRNA between Smad7 overexpression clone and the control groups whether stimulated by TGF-beta2 or not. The increase of p-Smad2 expression stimulated by TGF-beta2 was inhibited in Smad7 overexpression clone by Western blot assay. It only took 56.01% and 53.48% of control groups. In cellular immunofluorescence assay, the p-Smad2 positive cells in Smad7 overexpression clone only took 31.02% and 29.41% of control groups. Smad7 -HTFs could also abolish the increase of COL1A2 mRNA expression and PICP concentration stimulated by TGF-beta2. It is possible that Smad7 can block the signal transduction of TGF-beta by downregulating the expression of p-Smad2 in HTFs. However, it doesn't affect the expression of Smad2.
Chen
Jun Yi
JY
Department of ophthalmology, Eye & ENT Hospital of Fudan University, Shanghai 200031.
Sun
Xing Huai
XH
D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism*; D003239:Connective Tissue Cells; D005347:Fibroblasts / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D012333:RNA, Messenger / Q000378:metabolism*; D051899:Smad2 Protein / Q000235:genetics / Q000378:metabolism*; D051906:Smad7 Protein / Q000235:genetics*; D014162:Transfection
D024042:Collagen Type I; D012333:RNA, Messenger; C497782:SMAD2 protein, human; D051899:Smad2 Protein; D051906:Smad7 Protein