摘要目的 研究他克莫司对体外培养的人角质形成细胞蛋白酶活化受体2(PAR-2)表达及功能的影响.方法 10-9～10-5 mol/L他克莫司与人角质形成细胞共培养24 h后,采用半定量反转录-聚合酶链反应、免疫荧光和Western印迹分别检测人角质形成细胞PAR-2 mRNA和蛋白表达水平的变化,应用Fluo-4钙荧光探针检测不同浓度他克莫司对人角质形成细胞激活PAR-2后引起的细胞内钙离子浓度变化的影响,以不加他克莫司处理组为对照组.采用单因素方差分析比较各组人角质形成细胞PAR-2表达和细胞内钙离子浓度,LSD-t检验进行两两比较.结果 PAR-2在角质形成细胞的胞膜和胞质表达.角质形成细胞与10-9～10-5 mol/L他克莫司共培养24 h后,PAR-2 mRNA的表达水平与对照组比较均显著降低(均P<0.05),且与他克莫司浓度呈负相关(r=-0.962,P=0.009).免疫荧光和Western印迹显示,与对照组比较,10-5和10-6 mol/L他克莫司组PAR-2蛋白表达水平显著降低(均P<0.05),10-7 mol/L他克莫司组PAR-2蛋白表达水平亦降低(免疫荧光法P<0.05;Western印迹法P>0.05),但10-8和10-9 mol/L他克莫司组PAR-2表达水平变化无统计学意义(均P>0.05).共培养24 h后,10-5、10-6和10-7 mol/L他克莫司组PAR-2激活后细胞内钙离子峰浓度(峰值吸光度分别为1463±283、1455±270、1423±291)较对照组(1602±407)显著降低(t值分别为2.582、2.821、2.923,P值分别为0.032、0.022、0.019),10-8和10-9 mol/L他克莫司组(分别为1649±379、1633±415)变化无统计学意义(t值分别为0.846、0.462,P值分别为0.422、0.657).结论 他克莫司可抑制人角质形成细胞PAR-2的表达,并抑制PAR-2激动剂所引起的钙离子动员.

abstractsObjective To evaluate the in vitro effect of tacrolimus on the expression and function of protease-activated receptor 2(PAR-2)in cultured human keratinocytes. Methods After 24-hour co-culture of human keratinocytes with 10-9-10-5 mol/L tacrolimus, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR)was performed to determine the mRNA expression of PAR-2, immunofluorescence(IF)staining and Western blot analysis were performed to determine the protein expression of PAR-2 in the keratinocytes, and the fluorescent calcium probe fluo-4 was used to evaluate the effect of tacrolimus at different concentrations on the intracellular calcium concentration after the activation of PAR-2 in the keratinocytes. The group treated without tacrolimus served as control group. One-way analysis of variance was used to compare the PAR-2 expression and calcium concentration in the human keratinocytes in different groups, and least significant difference(LSD)-t test was carried out for multiple comparisons. Results PAR-2 was expressed on both the membrane and cytoplasm of keratinocytes. After 24-hour co-culture of keratinocytes with 10 - 9- 10 - 5 mol/L tacrolimus, the PAR-2 mRNA expression significantly decreased in these cells compared with the control group(all P < 0.05), and was negatively correlated with the tacrolimus concentration(r=-0.962, P=0.009). IF staining and Western blot analysis showed that the PAR-2 protein expression was significantly lower in the 10-5- and 10-6-mol/L tacrolimus groups than in the control group (both P < 0.05), and decreased to a certain extent in the 10-7-mol/L tacrolimus group(IF staining:P<0.05;Western blot analysis:P>0.05). No significant difference in thePAR-2 protein expression was observed between the 10-8-or 10-9-mol/L tacrolimus group and the control group(both P>0.05). After 24-hour co-culture, the peak concentration of intracellular calcium after PAR-2 activation was significantly lower in the 10-5-, 10-6- and 10-7-mol/L tacrolimus groups (peak absorbance:1463 ± 283, 1455 ± 270, 1423 ± 291 respectively)than in the control group(1602 ± 407;t=2.582, 2.821, 2.923, P=0.032, 0.022, 0.019, respectively), while there was no significant difference between the 10-8-or 10-9-mol/L tacrolimus group(1649 ± 379, 1633 ± 415 respectively)and the control group(t=0.846, 0.462, P=0.422, 0.657, respectively). Conclusion Tacrolimus can inhibit PAR-2 expression and suppress calcium mobilization induced by a PAR-2 agonist in keratinocytes.