TGF-β1调控miR-21抑制瘢痕疙瘩成纤维细胞凋亡的研究

Keloid is considered to be a kind of benign tumor.Fibroblast (FB) 's over proliferation and less apoptosis plays an important role in keloid formation, the mechanism is still unknown.TGF-β1is the primary cytokine driving fibrosis in various organs.Expression of TGF- β1 of keloid is significantly higher than that in normal skin, which can upregulates the expression of mature miR-21 by promoting the prosess of primary transcripts of miR-21(pri-miR-21)into precusor miR-21(pre-miR-21).MicroRNAs are small non-coding RNA molecules that regulate gene expression in the progress of proliferation, differentiation and apoptosis.Our group has confirmed that keloid has high expression of miR-21 compared to nomal skin and miR-21 can inhibit the apoptosis of FB. In the preliminary experiment, we found that:after transfection of antisense TGF- β1, miR-21 decreased significantly. Therefore, we hypothesized that 'TGF- β increased miR-21, miR-21 can cleave the target gene and block its expression, resulting in less apoptosis and over proliferation'. We intend to expounds that TGF-β1-induced gene regulation and apoptosis inhibition is partly mediated by miR-21 through some molecular techniques such as siRNA,Real-Time PCR, Western blot technology and so on.This study will provide new ideas and new targets for the pathogenesis and treatment of keloid.

成纤维细胞（FB）的异常增殖，细胞的凋亡减少，在瘢痕疙瘩形成中起重要作用，但机制不详。本课题组已经证实，瘢痕疙瘩中存在miR-21高表达，miR-21能促进瘢痕疙瘩的FB增殖。瘢痕疙瘩中的TGF-β1表达量显著高于正常皮肤，且可通过促进miR-21前体的成熟而上调miR-21的表达。在预实验中，我们发现FB转染了反义TGF-β1后，其miR-21表达明显降低。因此我们推测"瘢痕疙瘩中TGF-β1增高促进了miR-21表达，miR-21可通过与其靶基因（PTEN、PDCD4等肿瘤抑制因子）的特异性结合而阻断其表达，导致细胞增殖增多而凋亡减少"是瘢痕疙瘩发生的新机制。我们拟建立瘢痕疙瘩FB体外培养的模型，应用siRNA、转染、Real-time PCR、western blot等技术，在体外实验中证明我们的假说，此研究将为瘢痕疙瘩的发病机制及治疗提供新思路及新靶点。

微小RNA－21作为一种短链的非编码RNA，可能参与了瘢痕疙瘩发生发展的多种生物过程包括增殖、凋亡和分化的基因调节。瘢痕疙瘩中的TGF-β1表达量显著高于正常皮肤。本研究采用real－time PCR、western blot、siRNA转染、激光共聚焦、流式细胞仪和免疫荧光等技术，在体外细胞模型中首次观察到：miR－21的高表达与瘢痕疙瘩成纤维细胞的增殖、分化和凋亡密切相关；TGF-β1高表达可影响miR－21的表达，并且进一步使其靶基因发生表达变化，使靶基因所在的AKT信息传导通路发生改变，从而影响瘢痕疙瘩的凋亡、增殖和分化。上述结果证实了我们提出的“瘢痕疙瘩中 TGF-β1 增高促进了miR-21表达,miR-21可通过与其靶基因的特异性结合而阻断其表达,从而导致细胞增殖增多而凋亡减少”的假说，为瘢痕疙瘩的发病机制及治疗提供一个新的思路及新靶点。