摘要：
Objective To preliminarily evaluate the effect of levocetirizine hydrochloride at different concentrations on the growth of in vitro cultured human dermal papilla cells,and to explore its mechanism.Methods Human dermal papilla cells were divided into several groups to be cultured with Dulbecco's modified eagle medium (DMEM) containing 0 (control group),1,10,100,1 000,10 000 μg/L levocetirizine hydrochloride respectively for 48 hours.Immunofluorescence staining was performed to observe the growth of the dermal papilla cells,and methyl thiazolyl tetrazolium (MTT) assay to evaluate the proliferative activity of the dermal papilla cells.Real-time fluorescence-based quantitative PCR was conducted to measure the mRNA expression of cyclooxygenase 2 (COX-2),prostaglandin D2 synthase (PTGDS),prostaglandin E2 (PGE2),prostaglandin F2alpha (PGF2α),G protein-coupled receptor 44 (GPR44),protein kinase B (AKT) and glycogen synthase kinase 3β (GSK3β),and Western blot analysis to determine the protein expression of PTGDS.After 24-hour culture with DMEM containing levocetirizine hydrochloride at different concentrations,enzyme-linked immunosorbent assay (ELISA) was performed to detect the levels of prostaglandin D2 (PGD2) and PGD2R receptor in the culture supernatant of the human dermal papilla cells.Statistical analysis was carried out with SPSS 17.0 software using one-way analysis of variance for the comparison of the above indices among the groups,and least significant difference (LSD)-t test for multiple comparisons.Results Immunofluorescence staining showed that human dermal papilla cells grew well and reached over 90％ confluence in the 100 μg/L levocetirizine hydrochloride group.MTT assay revealed that there were significant differences in the proliferation rate among all the groups (F =42.22,P ＜ 0.05),and the proliferation rate was significantly higher in the 100 μg/L levocetirizine hydrochloride group (115.80％ ± 5.10％) than in the control group (100％,t =28.26,P ＜ 0.05).The mRNA expression(2-△△Ct) of COX-2,PGF2a,PTGDS,GPR44 and AKT all significantly differed among these groups (F =1.97,3.66,2.17,2.66 and 7.32 respectively,all P ＜ 0.05),while no significant difference in the mRNA expression of PGE2 and GSK3β was observed among these groups (F =0.87 and 1.19 respectively,both P ＞ 0.05).The 100 μg/L levocetirizine hydrochloride group showed significantly decreased mRNA expression of COX-2,PTGDS and GPR44 (0.84± 0.08,0.81±0.10 and 0.85 ± 0.09 respectively) compared with the control group (t =1.97,2.17 and 2.66 respectively,all P ＜ 0.05),but significantly increased mRNA expression of PGF2α and AKT (1.96 ± 0.25 and 1.74 ± 0.32 respectively) compared with the control group (t =3.66,7.32 respectively,both P ＜ 0.05).Moreover,the protein expression of PTGDS,PGD2 and PGD2R significantly differed among these groups (all P ＜ 0.05),and was significantly lower in the 100 μg/L levocetirizine hydrochloride group than in the control group (P ＜ 0.05).Conclusion Levocetirizine hydrochloride can promote the in vitro growth of human dermal papilla cells,likely by inhibiting the PGD2-GPR44 pathway and activating the AKT signal pathway.%目的 初步探讨不同浓度盐酸左西替利嗪对体外培养人毛乳头细胞(hDPC)生长的影响及机制.方法 将hDPC在含1、10、100、1 000、10 000 μg/L盐酸左西替利嗪的DMEM培养液中培养48 h,免疫荧光染色观察细胞的生长情况,MTT法检测细胞增殖活力,实时荧光定量PCR法检测环氧化酶2(COX-2)、前列腺素D2合酶(PTGDS)、前列腺素E2(PGE2)、前列腺素F2α(PGF2α)、G蛋白偶联受体44(GPR44)、蛋白激酶B(AKT)、糖原合成酶激酶3β(GSK3β)的mRNA表达水平;Western印迹法检测PTGDS蛋白水平.hDPC在含以上浓度盐酸左西替利嗪的DMEM培养液中培养24 h,酶联免疫吸附试验检测培养上清液中前列腺素D2(PGD2)和PGD2受体(PGD2R)的水平.以不加药物同等条件培养的hDPC为对照组.采用SPSS17.0软件进行单因素方差分析,组间两两比较使用LSD-t检验.结果 免疫荧光染色显示,100 μg/L组细胞生长良好,融合度超过90％.MTT法显示,不同浓度盐酸左西替利嗪组与对照组hDPC的增殖率差异有统计学意义(F=42.22,P＜0.05),100 μg/L组增殖率(115.80％±5.10％)高于对照组(100％,t=28.26,P＜0.05).不同浓度盐酸左西替利嗪组的COX-2、PGF2a、PTGDS、GPR44、AKTmRNA相对表达水平(以对照组表达为1)差异均有统计学意义(F值分别为1.97、3.66、2.17、2.66、7.32,P＜0.05),PGE2、GSK3β表达差异无统计学意义(F值分别为0.87、1.19,P＞0.05);100 μg/L组COX-2、PTGDS、GPR44 mRNA表达(0.84±0.08、0.81±0.10、0.85±0.09)低于对照组(t值分别为1.97、2.17、2.66,均P＜0.05),PGF2α、AKT的表达(1.96±0.25、1.74±0.32)高于对照组(t值分别为3.66、7.32,均P＜0.05).不同浓度盐酸左西替利嗪组PTGDS、PGD2、PGD2R水平差异均有统计学意义(P＜0.05),100 μg/L组PTGDS、PGD2、PGD2R蛋白水平均低于对照组(P＜o.05).结论 盐酸左西替利嗪可能通过抑制PGD2-GPR44通路,激活AKT信号通路,促进体外培养人毛乳头细胞的生长.
