Gremlin对百草枯诱导肺成纤维细胞胶原合成的影响

The effects of Gremlin on collagen expression induced by paraquat in lung fibroblasts

[论文特点介绍]Gremlin是骨形态发生蛋白（BMP）拈抗剂，在纤维化病理状态下可见表达，是否参与纤维化发病并无准确定论。本研究利用Gremlin多抗中和Gremlin的生物作用，通过体外正常肺成纤维细胞与百草枯共培养，对比加入不同浓度的Gremlin抗体，观察细胞形态学变化并检测α-SMA、胶原Ⅰ、胶原Ⅲ蛋白表达。探讨百草枯中毒环境中成纤维细胞Gremlin蛋白表达水平以及内源性Gremlin对百草枯介导成纤维细胞胶原合成影响。

Background and objective The patient with paraquat intoxation can develop to pulmonary fibrosis after acute stage,which is high mortality and mutilation. The pathogenesy of paraquat induced pulmonary fibrosis is primarily unknown. There has no specific treatment options with significant effect on patient survival at present. Gremlin, secreted antagonists of bone morphogenesis proteins, have been shown to over express in fibrotic disease, however, the profibrotic role of Gremlin has not been clarified so far. The study suggests that paraquat is characterized by resulting in fibrosis directly and induces Gremlin expression. The endogenous Gremlin plays an important role in collagen production induced by paraquat. Methods Primary lung fibroblasts isolated from BALB/C mouse were cultured, then, identified with cell shape and immunocytochemistry. The fibroblasts were divided into 5 groups, control group： the cells treated with medium. PQ group： the cells treated with 25 μM PQ in the medium. Gremlin Ab group： the cells treated with Gremlin Ab 5 μg/ml in the medium. PQ＋ Gremlin Ab group： the cells treated with ：25 M PQ and Gremlin Ab in the medium. After 12 h, Immunohistochemistry （IHC） was used to compare the production of collagen type Ⅰ and type Ⅲ an α-SMA. Results （1）The primary cells showed round nucelus and fusiform shape under microscope. Immunoetyochemistryassay showed that cultured lung cells had positive staining of vimentin, but negative of keratin. These data proved that the cultured cells were lung fibroblasts.（2）The cultured cell incubated with 25μM paraquat over expressed gremlin by IHC,in contrast,normal fibroblasts expressed no gremlin（P〈 0.05）.（3） At the protein level, expression of α-SMA, Col Ⅰ and Col Ⅲ in cultured fibroblasts was measured by immunocytochemical method. The results showed paraquat induced fibroblasts expression of α-SMA, Col Ⅰ, Col Ⅲ, and Gremlin Ab inhibited the effects of paraquat on the expression of α-SMA, Col Ⅰ, Col Ⅲ in fibroblasts in dose-independent manner（P〈0.05）. Conclusions paraquat stimulated fibroblasts expression of endogenous gremlin and collagen production. To give gremlin affinity purified polyclonal Ab inhibit collagen synthesis induced by paraquat.