转染Smad7基因的人眼球筋膜囊成纤维细胞Ⅰ型胶原蛋白及纤维连结蛋白表达的改变  

Changes of type Ⅰ collagen and fibronectin expressions on cultured human Tenon＇s capsule fibroblasts transfected with Smad 7 vector

作　　者：陈君毅[1] 孙兴怀[1] 

机构地区：[1]复旦大学附属眼耳鼻喉科医院眼科,上海200031

出　　处：《中华眼科杂志》2007年第2期124-128,共5页Chinese Journal of Ophthalmology

摘　　要：目的观察转染Smad7基因的人眼球筋膜囊成纤维细胞（HTFs）Ⅰ型胶原蛋白和纤维连结蛋白表达的改变。方法用Nucleofector^TM核酸转染仪将含有Smad7的真核表达质粒转染至HTFs。采用实时定量逆转录聚合酶链反应法检测Ⅰ型胶原蛋白及纤维连结蛋白mRNA表达的改变。采用放射免疫法检测Ⅰ型前胶原羧端肽原（PICP）在培养液中浓度的改变，检测经转化生长因子β2（TGF-β2，10ng／ml）作用后以上指标的改变。以未转染HTFs和转染空质粒的HTFs作为对照。结果成功将含有Smad7基因的质粒转染至HTFs，并证明其Smad7基因mRNA表达明显升高。转染Smad7的HTFsⅠ型胶原蛋白mRNA表达率为（89．53±9．37）％，分别是对照组HTFs的40．47％和pCMV5-HTF8的37．94％。培养液中PICP浓度为（15．29±2．18）ug／L，分别是对照组HTFs的52．10％和pCMV5-HTFs的57．35％。转染Smad7的HTFs能够明显拮抗由TGF-β2作用引起的Ⅰ型胶原蛋白mRNA表达和PICP浓度升高（P〈0．01）；无论是否有TGF-β2作用，转染Smad7的HITs纤维连结蛋白mRNA的表达与对照组相比，差异均无统计学意义（P〉0．05）。结论Smad7能降低HTFsⅠ型胶原蛋白mRNA表达和Ⅰ型胶原蛋白的合成，但对纤维连结蛋白mRNA表达无明显作用。Objective To investigate type Ⅰ collagen and fibronectin expressions in the cultured human Tenon＇s capsular fibroblasts （HTFs） transfected with Smad 7 vector and to elucidate the possibility of Smad 7 in blocking tissue fibrosis after filtration surgery. Methods Nucleofector^TM was used to transfect Smad 7 vector into HTFs. The expressions of α2-type Ⅰ precollagen （ COL1 A2 ） mRNA and fibronectin mRNA were detected by reverse transcription real-time quantitative polymerase chain reaction （real-time RT- PCR）. The concentration of carboxyterminal propeptide of type Ⅰ precollagen （PICP） in culture media was detected by radioimmunoassay. The above mentioned experiments were also detected before and after HTFs stimulated by TGF-β2 （ 10 ng/ml）. HTFs and vector-HTFs were used as control groups. Results Smad 7 was successfully transfected into HTFs evidenced by Smed 7 over expression. The expression of COL1A2 mRNA in Smad 7-HTFs was decreased 40. 47% and 37.94%, respectively, when compared with the control HTFs and pCMVS-HTFs group. The PICP concentration of Smad 7-HTFs in the culture medium was decreased 52. 10% and 57.35%, respectively, comparing to the two control groups. Furthermore, Smad 7- HTFs attenuated the increase of COL1A2 mRNA expression and PICP concentration of HTFs stimulated by TGF-β2. There was no difference in the fibronectin mRNA expressions between Smad 7-HTFs and the two control groups. Conclusions Smed 7 reduces the expression of COL1A2 mRNA and the synthesis of type Ⅰ collagen, but has no effect on fibronectin mRNA expression, which may imply its ability to inhibit tissue fibrosis after filtration surgery.

关 键 词：转染 眼 成纤维细胞 SMAD 7蛋白 胶原Ⅰ型 纤连蛋白类 

分 类 号：R686[医药卫生—骨科学]