Wnt/Notch信号通路网在瘢痕疙瘩中的研究

Keloids are benign dermal tumor. Now there are no effective ways for preventing and curing keloids because the definite pathogenesy is still unknown. So far it is revealed that the key cells, keloid fibroblasts, stimulate type Ⅰcollagens over expression is the main pathomechanism. In the early experiment, we confirmed that keloid-derived mesenchymal-like stem cells are existence, and we succeed to cultivate and identify the cells and discover that they can give rise to keoid fibroblasts in vitro. Moreover, GSK-3β is low expression in keloid-derived mesenchymal-like stem cells. As all know, Wnt and Notch signal pathway take an important part in the generation and differentiation of turmor stem cells. According to all reasearches, we presume that GSK-3β can media Wnt/Notch cross-talk, and regulated the formation of keloid. In this study, we will take some molecular biology technique, such as RT-PCR, western blotting, immunofluorescence, ChIP、GST pull-down、RNAi and EMSA, to research systemically the realationship of the expression of GSK-3β, and to observe the biological behaviour of keloid under GSK-3β control in vivo and vitro. So that we could approach the mechanism of GSK-3β mediating Wnt/Notch cross-talk. So we could provide a new target for preventing and curing keloids, moreover, provide a new clue and theory.

瘢痕疙瘩是皮肤的良性肿瘤，其发病机制未明，临床上尚无有效治疗手段。主要病理机制为其关键效应细胞- - 瘢痕疙瘩成纤维细胞生成过度的Ⅰ型胶原。我们前期工作证实了瘢痕疙瘩中存在瘢痕疙瘩干细胞；体外成功培养了瘢痕疙瘩干细胞，发现其可分化为瘢痕疙瘩成纤维细胞；糖原合成酶激酶-3β（GSK-3β）在瘢痕疙瘩干细胞中低表达。已知Wnt和Notch信号通路在肿瘤干细胞的增殖和分化中起着重要作用。结合前人研究，我们推测GSK-3β通过介导Wnt/Notch串话，调控瘢痕疙瘩形成。本课题将采用RT-PCR、蛋白印迹、免疫荧光、腺病毒重组、ChIP、GST pull-down、RNAi以及EMSA等技术，系统研究GSK-3β表达与Wnt/Notch信号通路的关系，在体和离体模型上观察GSK-3β对瘢痕疙瘩生物行为的影响，探讨GSK-3β介导Wnt/Notch串话的机制，为防治瘢痕疙瘩提供新的靶点和新的线索与理论

瘢痕疙瘩的发病机制目前仍不清楚。瘢痕疙瘩干细胞的研究，有可能从根本上阐明瘢痕疙瘩的发病机制。我们的研究证实了：1、完成瘢痕疙瘩干细胞的体外培养和鉴定。通过流式细胞仪检测，瘢痕疙瘩干细胞表达CD73，CD90等表面抗原，而不表达CD45等分子；同时，进一步将该细胞传代扩增，证实其具有一定的传代能力而不会分化；另外，成功构建了裸鼠瘢痕模型。2、在体外实验中，通过以正常成纤维细胞作为对照，对瘢痕疙瘩干细胞及正常成免疫荧光染色、实时荧光定量PCR以及Western Blotting实验，发现了在Wnt和Notch信号通路主要成员中GSK-3β、β-catenin、Jagged1、Notch1等都有表达上的差异，提示GSK-3β、β-catenin、Jagged1、Notch1等有可能涉及到瘢痕疙瘩的增生发展。3、过表达GSK-3β后，检测过表达成功，再检测Wnt和Notch信号通路主要成员中wnt5a、β-catenin、Jagged1，发现wnt5a、β-catenin、Jagged1表达上调，提示GSK-3β可能同时介导了Wnt和Notch两条信号通路的串话。进一步将分析GSK-3β与Notch信号通路的具体结合位点。目前实验还在正进行，即用EMSA证实GSK-3β的结合序列是否能够与Notch1结合。4、进一步利用有限的经费，检测了microRNA和circRNA在瘢痕疙瘩中表达情况，与正常皮肤组织相比，瘢痕疙瘩中发现了2591个差异microRNA，18277个差异circRNA，并且做了功能富集分析，初步筛选出与瘢痕疙瘩报道的功能相关RNA，为下一步的研究提供了基础。5、课题组前期研究发现，TGF-β1/Smads 信号转导通路最重要的负反馈调节信号分子Smad7表达明显下调，瘢痕疙瘩中TIEG1直接调控Smad7 基因表达。进一步研究发现，瘢痕疙瘩中TIEG1的降解是由泛素系统决定的。该研究结果已被J Invest Dermatol接收。