过表达TRPM8在SCN5A错义突变后对肥大细胞功能的影响

Effect of TRPM8 overexpression on mast cellular functions after missense mutation of SCN5A

目的探究过表达瞬时受体电位阳离子通道M8(TRPM8)在心脏钠通道基因(SCN5A)错义突变后对肥大细胞功能的影响。方法构建TRPM8基因过表达载体及SCN5A干扰载体并分别转染入人肥大细胞HMC-1中,转染成功后用CCK8检测细胞增殖能力,qPCR、Western blot法检测TRPM8与SCN5A的表达情况,中性红染色计算肥大细胞脱颗粒百分率,比色法测定β-氨基己糖苷酶释放率。结果TRPM8基因过表达及SCN5A干扰载体成功构建;与空白对照组相比,过表达TRPM8与干扰SCN5A后均抑制了HMC-1细胞的增殖能力,过表达TRPM8对HMC-1细胞增殖能力的抑制作用低于干扰SCN5A组(P<0.05);与干扰SCN5A组相比,干扰SCN5A的同时过表达TRPM8后对HMC-1细胞增殖能力的抑制作用有所降低(P<0.05);与空白对照组相比,过表达TRPM8降低HMC-1细胞脱颗粒反应和β-氨基己糖苷酶释放率,干扰SCN5A后增强了HMC-1细胞脱颗粒反应和β-氨基己糖苷酶释放率(P<0.05);与干扰SCN5A后相比,在干扰SCN5A的同时过表达TRPM8可降低HMC-1细胞脱颗粒反应和β-氨基己糖苷酶释放率(P<0.05);干扰SCN5A后可下调HMC-1细胞中TRPM8的表达(P<0.05)。结论TRPM8可以抑制SCN5A错义突变后对肥大细胞的激活作用,并可能通过调控SCN5A来减缓肠易激综合征患者疼痛等不适症状。

Objective To investigate the effect of overexpression of transient receptor potential melastatin 8(TRPM8)on mast cellular functions after missense mutation of SCN5A.Methods The vector overexpressing TRPM8 and the vector silencing SCN5A were constructed and transfected into human mast cell HMC-1,respectively.After successful transfection,cell proliferation was detected by CCK8 assay,and the expressions of TRPM8 and SCN5A were detected by qPCR and Western blot.Neutral red staining was performed to calculate the percentage of mast cell degranulation.The release rate ofβ-amino-hexosidase was determined by colorimetry.Results TRPM8 gene overexpression vector and SCN5A interference vector were successfully constructed.When compared with blank control group,TRPM8 overexpression and SCN5A interference inhibited HMC-1 cell proliferation.The inhibition by of TRPM8 overexpression on HMC-1 cell proliferation was weaker than that by SCN5A interference(P<0.05).When compared with SCN5A interference group,TRPM8 overexpression while interfering with SCN5A reduced the inhibitory effect on HMC-1 cell proliferation(P<0.05).When compared with blank control group,TRPM8 overexpression attenuated the degranulation reaction andβ-aminocaprosidase release rate of HMC-1 cells,while SCN5A interference enhanced the degranulation reaction andβ-aminocaprosidase release rate of HMC-1 cells(P<0.05).When compared with SCN5A interference,TRPM8 overexpression during SCN5A interference decreased the degranulation reaction andβ-amino-hexosidase release rate of HMC-1 cells(P<0.05).SCN5A interference downregulated TRPM8 expression in HMC-1 cells(P<0.05).Conclusion TRPM8 can inhibit the activation effect of SCN5A missense mutations on mast cells and may alleviate discomfort symptoms such as pain in patients with irritable bowel syndrome by regulating SCN5A.