ROCK抑制剂Y-27632促进人角质形成细胞增殖与调控衰老

ROCK inhibitor Y-27632 promotes proliferation of human keratinocytes and inhibits its senescence

背景:促进角质形成细胞体外培养增殖能力对皮肤生物学和皮肤相关疾病发病机制的研究具有重要意义。目的:观察Rho激酶抑制剂Y-27632对角质形成细胞增殖与衰老的影响。方法:分离培养人角质形成细胞,通过形态鉴定观察Y-27632(10μmol/L)对角质形成细胞形态的影响;利用群体倍增速率估算Y-27632对角质形成细胞增殖的影响;分别在角质形成细胞第0,5代,通过β-半乳糖苷酶细胞染色方法检测Y-27632对细胞衰老的作用;进一步通过MTT法和BrdU细胞增殖检测试剂盒检测细胞增殖情况。结果与结论:人角质形成细胞为大小均一的铺路石样细胞,且在早期具有较强的增殖能力,随着细胞传代,增殖能力降低,且形态变为扁平的,胞浆增大的特点;Y-27632显著提高了细胞的群体倍增速率,通过β-半乳糖苷酶细胞染色显示,Y-27632降低了阳性细胞数,进一步通过MTT法和BrdU细胞增殖检测法得出,Y-27632促进细胞体外增殖。结果说明,人角质形成细胞体外过程中,Y-27632的加入不仅能够抑制细胞衰老,同时促进其增殖,为角质形成细胞体外扩增培养与研究提供了新思路。

BACKGROUND： Facilitating the proliferation of keratinocytes in vitro is of great significance for the studies on dermatobiology and the pathogenesis of skin-related diseases. OBJECTIVE： To investigate the effects of Rho kinase inhibitor Y-27632 on the proliferation and senescence of keratinocytes. METHODS： Human keratinocytes were successfully isolated and the potential effect of Y-27632（10 μmol/L） on the human keratinocytemorphology was investigated. The effect of Y-27632 on the proliferation of keratinocytes was investigated by population doubling curve; β-galactosidase staining was performed at P0 and P5 cells respectively to show the effect of Y-27632 on the senescence of keratinocytes. Finally, the proliferation of keratinocytes was detected by MTT assay and BrdU kit simultaneously. RESULTS AND CONCLUSION： Human keratinocytes were of cobble-stone morphology and equal size, which kept great proliferation capacity at early stage. With the continuous passage of human keratinocytes, their proliferation capacity was declined gradually, showing a flat morphology and an enlarged cytoplasmic volume. The population doubling of human keratinocytes was increased significantly in the presence of Y-27632, and according to the β-galactosidase staining results, Y-27632 decreased the number of positive cells. Furthermore, the results of MTT assay and BrdU kit indicated that Y-27632 facilitated the proliferation of keratinocytes obviously in vitro. That is to say, when the human keratinocytes are cultured in the presence of Y-27632, the senescence of keratinocytes is inhibited. Meanwhile, the proliferation of keratinocytes is significantly increased, and our data shed new insight on the in vitro proliferation of human keratinocytes in future studies.