Chen Jun Yi, Sun Xing Huai
Department of ophthalmology, Eye & ENT Hospital of Fudan University, Shanghai 200031.
Fen Zi Xi Bao Sheng Wu Xue Bao. 2006 Jun;39(3):265-70.
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.
本研究旨在探讨转染Smad7载体的培养人Tenon囊成纤维细胞（HTF）中Smad2、磷酸化Smad2（p-Smad2）和I型胶原蛋白的表达情况，并阐明Smad7在滤过性手术后阻断组织纤维化的机制。采用核转染技术将Smad7载体转染至HTF。随后使用逆转录实时定量聚合酶链反应（实时RT-PCR）检测Smad7 mRNA表达水平。通过实时RT-PCR、蛋白质免疫印迹法、细胞免疫荧光测定法和放射免疫测定法测定HA-探针融合蛋白、Smad2、p-Smad2、α2-I型前胶原（COL1A2）mRNA和I型前胶原羧基末端前肽（PICP）的表达。在HTF受到TGF-β2（10 ng/ml）刺激后，也对上述指标进行检测。将HTF和载体-HTF作为对照组。成功建立了Smad7过表达克隆。实时RT-PCR结果证明，无论是否受到TGF-β2刺激，Smad7过表达克隆与对照组之间Smad2 mRNA均无差异。蛋白质免疫印迹法检测显示，Smad7过表达克隆中TGF-β2刺激引起的p-Smad2表达增加受到抑制。其仅为对照组的56.01%和53.48%。细胞免疫荧光测定法显示，Smad7过表达克隆中p-Smad2阳性细胞仅为对照组的31.02%和29.41%。Smad7-HTF也可消除TGF-β2刺激引起的COL1A2 mRNA表达增加和PICP浓度升高。Smad7可能通过下调HTF中p-Smad2的表达来阻断TGF-β的信号转导。然而，它并不影响Smad2的表达。
