蛋白酶激活受体2在人角质形成细胞中的表达及功能测定

目的 分析人皮肤角质形成细胞中蛋白酶激活受体2（PAR2） 的表达及其生物学功能，探讨其在皮肤屏障中的作用。方法 以体外培养的人皮肤角质形成细胞原代细胞或细胞系N//TERT 细胞为研究对象，通过免疫荧光染色及蛋白印迹分析细胞中PAR2的表达及分布特点，采用荧光标记的钙流释放法测定PAR2的生物学功能，利用PAR2 的天然底物胰蛋白酶或其激活肽、对照肽及拮抗肽诱导PAR2的活化，观察不同作用底物诱导PAR2活化的特点。 结果 在正常培养条件下，PAR2在皮肤角质形成细胞中呈中低水平表达，不同的培养基组成及培养时间对PAR2的表达水平无明显影响。PAR2的作用底物诱导细胞的钙流释放具有时间及浓度依赖性特点，其中使用50 ～ 250 nmol/L胰蛋白酶作用于角质形成细胞后，可以诱导细胞内钙流释放；使用75 ～ 250 μmol/L PAR2特异性激活肽PAR2-AP （H2N-SLIGKV-COOH）处理角质形成细胞后，即可最大程度地诱发PAR2的活化。而在相同浓度PAR2的拮抗肽PAR2-ANTAP （H2N-FSLLRY-COOH）作用下，PAR2的作用底物所诱导的细胞钙流释放明显受到抑制。分析比较相关荧光强度发现，PAR2激活底物诱导的细胞内钙流释放可以在瞬间（0 ～ 250 s）完成，以50 s的作用时间点细胞内钙流释放强度最大。此外，PAR2的激活可以显著增加皮肤角质形成细胞ERK蛋白的磷酸化水平，从而激发细胞内MAPK信号转导通路。结论 皮肤角质形成细胞表面表达有PAR2受体，该受体活化受胰蛋白酶及PAR2的特异性激活肽调控，PAR2的活化可影响角质形成细胞的生理功能，诱导细胞内钙流释放。

Analysis of protease-activated receptor 2 expression and function in cultured human keratinocytes

Objective To assess the expression pattern of protease-activated receptor 2 （PAR2） in human keratinocytes and to characterize its biological functions in the regulation of skin barrier. Methods Primary human keratinocytes and human N//TERT keratinocytes were used as the subject of this study. The expression and distribution of PAR2 in the keratinocytes were analyzed by using immunoflorescence staining and Western blot. Two different PAR2 agonists, trypsin and a PAR2-activating peptide（AP）, as well as a PAR2-antagonistic peptide （H2N-FSLLRY-COOH） and a control peptide were used to induce the activation of PAR2 in the keratinocytes. Then, a fluorescence-based calcium mobilization assay was performed to evaluate the biological function of PAR2. Data were statistically analyzed by one-factor analysis of variance. Results Under normal culture conditions, PAR2 was weakly expressed in keratinocytes, and the expression was unaffected by culture medium composition or culture duration. Calcium mobilization was induced by trypsin of 50－250 nmol/L and the PAR2-activating peptide in a dose- and time-dependent pattern. The maximal activation of PAR2 was observed in keratinocytes treated with the PAR2 agonist HAN-SLIGKV-COOH of 75－250 μmol/L. The PAR2-antagonistic peptide （H2N-FSLLRY-COOH） obviously suppressed the increase in calcium mobilization induced by trypsin, while the control peptide PAR-RAP showed no inductive effect on the PAR2 activation based on the absence of calcium mobilization. The substrate-induced calcium release was complete within 250 seconds, and peaked at 50 seconds after the initial trypsin or PAR-AP stimulation. Moreover, the activation of PAR2 was accompanied by an increase in ERK phosphorylation and elicitation of MAPK signaling pathway in keratinocytes. Conclusions Human keratinocytes positively express PAR2, which can be activated by trypsin and PAR2-activating peptides, and the activation of PAR2 may influence the physiological function of keratinocytes by inducing intracellular calcium release