miRNA-21调控ASCs失巢凋亡促进瘢痕创面汗腺再生的分子机制研究

Some area of skin without sweat glands have seriously influenced on the function of perspiration and caused the patients not to regulate their body temperature effctively,which has resulted in the severve and endless suffering. Thus,it is important significance to promote the regeneration of sweat gland for improving the quality of patients'living.Because adiposed derived stem cells(ASCs) can be induced to differentiate towards sweat gland epithelial cells without the challenge of ethics and difficulty in obtaining cells,they have become the most excellent kind of stem cells in the aspect of sweat gland regeneration so far. According to the recent studies,it has been proved most of ASCs may be anoikis during the course of differentiation into sweat gland epithelial cells and slow the efficiency of sweat gland regeneration.We recently found that miRNA-21 could be related to anoikis of ASCs.However,the detailed molecular mechanism have be unknown. To analyze the molecular network concerning the regulation of ASCs anoikis by miRNA-21 for improving sweat gland regeneration,we will firstly check the different expression of apoptotic related genes between ASCs and sweat gland epithelial cells derived from ASCs,then we will carry out the luciferase report assay system for elucidating the direct regulation of miRNA-21 on apototic related genes mentioned above.Both simulacrum and repressor of miRNA-21 will be respectively constructed and then be transfected into both ASCs and sweat gland epithelial cells derived from ASCs.On the basis of those,we in ture analyze the detailed molecular mechanism concerning the action that miRNA-21 and its target genes relevant to apoptosis take patrs in the regulation of ASCs anoikis;and through the above experiment we can evaluate the influence of miRNA-21 on differentiation of ASCs towards sweat gland epithelial cells.Based on the in vitro experiments mentioned above,we will perform the animal experiments.The nude mice model about skin wound surface will be made.Both ASCs and sweat gland epithelial cells derived from ASCs will be transplanted into the wound surface of animal model.On the basis of nude mice model,we can analyze the molecular mechanism on the regulation of ASCs anoikis by miRNA-21 in vivo experiments, which furtherly help to elucidate the effects of miRNA-21 on the regeneration of scar surface's sweat gland. This study will thoroughly solve the key scientific problems that slowed down the efficiency of sweat gland regeneration and the theoretical conclusions of those studies will provide the novel target points and theoretical basis for controlling ASCs differentiation toward swear gland epithelial cells and make sweat gland regenration more effective and efficient on skin wound and scar surface.

大面积瘢痕创面汗腺缺失影响体温调节功能带给患者无尽痛苦，脂肪干细胞（ASCs）可分化为汗腺上皮细胞且容易获取，从而成为汗腺再生的理想干细胞；但ASCs在定向诱导中容易发生失巢凋亡进而抑制汗腺再生,因此有效控制ASCs失巢凋亡成为促进汗腺再生的关键。前期证实miRNA-21参与调控ASCs失巢凋亡,但机制不详。本研究以此为切入点,首先体外筛选ASCs凋亡相关基因并验证miRNA-21对其调节作用；然后构建miRNA-21模拟物和阻遏物并转染ASCs及其分化的汗腺上皮细胞；分析miRNA-21及其靶基因调控ASCs失巢凋亡的分子机制进而阐明miRNA-21对ASCs诱导分化的影响，最后制备动物模型体内验证miRNA-21调控ASCs失巢凋亡的作用机制并评价该机制对创面汗腺再生的影响。本研究将消除制约汗腺再生的瓶颈问题，为汗腺再生提供新的基因调控靶点，具有十分重要的理论意义和潜在的临床应用价值。

大面积瘢痕创面汗腺缺失影响体温调节功能带给患者无尽痛苦，脂肪干细胞（ASCs）可分化为汗腺上皮细胞且容易获取，从而成为汗腺再生的理想干细胞；但ASCs在定向诱导中容易发生失巢凋亡进而抑制汗腺再生,因此有效控制ASCs失巢凋亡成为促进汗腺再生的关键。前期证实miRNA-21参与调控ASCs失巢凋亡,但机制不详。本研究以此为切入点,完成体外分离培养汗腺组织，体外获取人汗腺上皮细胞并传代培养。完成EDA-A1基因转染人脂肪干细胞,观察和检测转染前后脂肪干细胞形态及表面抗原.初步探索了应用EGF和Transwell共培养诱导脂肪干细胞向汗腺上皮细胞分化,证实经诱导后的脂肪干细胞形态及表型发生明显变化,有向汗腺上皮细胞分化的趋势。完成了miRNA21转染人脂肪干细胞(ADSCs)，分析了miRNA-21及其靶基因调控ASCs失巢凋亡的分子机制进而阐明miRNA-21对ASCs诱导分化的影响，课题组发现MiRNA-21在瘢痕创面愈合中上调,MiRNA-21不仅调控瘢痕成纤维细胞的增殖,还通过线粒体凋亡途径影响着成纤维细胞的凋亡.。本研究将消除制约汗腺再生的瓶颈问题，为汗腺再生提供新的基因调控靶点，具有十分重要的理论意义和潜在的临床应用价值。本研究已发表论文6篇，其中SCI收录4篇，培养硕士研究生11名，博士研究生2名。