大麻素2型受体激动剂JWH133对人永生化角质形成细胞体外增殖以及细胞因子表达影响的研究

Effects of cannabinoid type 2 receptor agonist JWH133 on proliferation of HaCaT cells and the expression of cytokines in vitro

目的探讨JWH133对人永生化角质形成细胞CHaCaT细胞)体外增殖以及经肿瘤坏死因子(TNF)-α诱导后细胞因子表达的影响。方法首先用不同浓度JWH133(15、30、60μmol/L)分别作用于HaCaT细胞。采用CCK-8法检测JWH133作用12、24、36 h后对HaCaT细胞体外增殖的影响,流式细胞仪检测JWH133对HaCaT细胞凋亡率的影响。其次采用抗体芯片检测技术分别检测20 ng/ml TNF-α组、20 ng/ml TNF-α.+30μmol/L JWH133组中细胞因子表达的变化,并采用双抗体夹心酶联免疫吸附试验CELISA)法进行定量分析,以验证抗体芯片检测结果的可靠性。结果15、30、60μmol/L JWH133作用12、24、36 h后对HaCaT细胞体外增殖均有不同程度的抑制作用。随着时间延长和JWH133浓度的增加,JWH133对HaCaT细胞体外增殖的抑制作用均逐渐增强(F=187.1、2678.9,P<0.05)。60μmol/LJWH133作用24h后,凋亡率由(4.34±0.07)%(阴性组〕增加至(21.77±0.40)%(JWH133组)(P<0.05)。20 ng/ml TNF-α能够诱导HaCaT细胞不同程度增加白细胞介素19CIL”19)、IL-22、IL-23等细胞因子的高表达,而30μmol/L JWH133处理后可降低上述细胞因子的表达。结论大麻素2型受体激动剂JWH133可抑制HaCaT细胞体外增殖、促进凋亡,且能够抑制TNF-α诱导的HaCaT细胞中一些重要免疫分子的表达。

Objective To evaluate the effects of cannabinoid type 2 receptor agonist JWH133 on the proliferation of human immortalized keratinocytes(HaCaT cells)in vitro and expression of cytokines induced by TNF-α.Methods First,HaCaT cells cultured in vitro were divided into 5 groups and treated with JWH133 at different concentrations of 15,30 and 60μmol/L(15,30 and 60(μmol/L JWH133 groups),DMEM containing the same volume of DMSO as in 60μmol/L JWH133 group(vehicle group),DMEM(negative group),respectively.Cell counting kit-8 treatment assay was performed to assess in vitro the cellular proliferative activity after 12-,24-,36-hour treatment,and the apoptosis rate was detected by flow cytometry.Then,the changes of cytokines expression in negative group,20 ng/ml TNF-αgroup and 20 ng/ml TNF-αcombined with 30μmol/L JWH 133 group were detected by antibody array,finally,the method of ELISA for quantitative analysis was adopted to check the reliability of the data obtained by antibody array.Results After treatment with JWH 133 of 15-60μmol/L for 12?36 hours,the proliferation of HaCaT cells was significantly inhibited to different extents compared with negative group(all P<0.05),and the inhibition rate significantly increased with the increase of treatment duration and concentrations of JWH 133(F=187.1,2678.9,respectively,P<0.05).Under the treatment with JWH 133 of 60 pmol/L for 24 hours,the apoptosis rate of HaCaT cells was increased from(4.34±0.07)%to(21.77±0.40)%(P<0.05).20 ng/ml TNF-αcould induce the high expression of interleukin 19(IL-19),IL-22,IL-23 and other cytokines in HaCaT cells to different extents,but the expression of these cytokines was decreased after being treated with 30μmol/L JWH133.Conclusion JWH133,a cannabinoid type 2 receptor agonist,can inhibit proliferation and induce apoptosis of HaCaT cells in vitro and inhibit the expression of some important immune molecules.