硫化氢对大鼠主动脉平滑肌细胞三磷酸腺苷敏感的钾离子通道基因表达的影响

Effects of Hydrogen Sulfide on Gene Expression of Adenosine Triphosphate-Sensitive Potassium Channel of Aortic Vascular Smooth Muscle Cells in Rats

目的研究硫化氢(H2S)对大鼠主动脉平滑肌细胞(ASMC)三磷酸腺苷(ATP)敏感的钾离子通道(KATP)内向整流性钾通道亚基(Kir6.1)和磺脲类受体亚基(SUR2B)基因表达的影响。方法采用SD雄性大鼠,贴块法原代培养ASMC,实验用4、5代细胞,分为H2S实验组和阴性对照组,分别同步化24h后,在H2S实验组中分别加入终浓度为10-5、10-4及10-3mol/L的H2S供体硫氢化钠,阴性对照组加入等体积的9g/L盐水,H2S实验组和阴性对照组继续处理24h。采用形态学观察和细胞免疫化学方法鉴定ASMC及纯度,采用细胞免疫化学方法鉴定KATP的SUR2B亚基和Kir6.1亚基在平滑肌细胞的表达定位;采用实时荧光定量PCR法分别检测各组细胞KATP24h后Kir6.1亚基和SUR2B亚基mRNA表达水平。结果镜下可见ASMC呈典型峰-谷状生长,经抗α平滑肌肌动蛋白单克隆抗体对培养的第4代ASMC进行免疫细胞化学染色,97%以上的细胞为阳性。应用SUR2B和Kir6.1纯化多克隆抗体对ASMC进行免疫细胞化学染色,发现SUR2B亚基和Kir6.1亚基分别在ASMC胞膜和胞质有阳性表达,而胞核无阳性表达;H2S实验组KATPSUR2B mRNA和Kir6.1mRNA表达水平较阴性对照组KATPSUR2B mRNA和Kir6.1mRNA表达水平显著增加,而且呈剂量依赖性。结论H2S能对KATP通道基因水平进行调节,H2S供体能使大鼠ASMCKATP通道SUR2B亚基和Kir6.1亚基mRNA表达上调,并在10-5～10-3mol/L呈质量浓度依赖性。

Objective To investigate the effect of hydrogen sulfide （H2S） on gene expression of adenosine triphosphate（ATP） -sensi- tive K ＋ channel （ KATP ） of aortic smooth muscle cells （ASMC） in rat. Methods Male Sprague - Dawley （SD） rats were used in the study. The original generation cells were obtained by modified tissue piece inoculation. The passaged cells were used. The ASMC were divided into 2 groups：H2 S group of different dosages and control group. The contents of sodium hydrosulfide in the culture media of H2 S group were 10^-5, 10^-4 and 10^-3 mol/L respectively,and the same volume of normal saline was added to control group. Each group was treated for 24 hours. Morphology of the cells was observed by inverted microscope and identified by immunohistochemical method employing α- smooth muscle - The expressions of SUPC2B and Kilt. 1 were identified by immunohistochemical SP technology. The SUR2B mRNA and Kirt. 1 mRNA levels were assayed by real time fluorescence relative quantitative polymerase chain reaction. Results The passaged cells developed typical growth pattern peak and valley and the 97th percent of the cells expressed the smooth muscle specific differentiation marker ct - smooth muscle - actm. SUR2B and Kirt. 1 could be detected in ASMC by immunohistochemical technology. They were both located at cytoplasmic and cy- tomembrane but not in at nucleus. Compared with control group, SUR2B mRNA and Kit6.1 mRNA levels in H2S groups were higher than those in control group in a dosage -dependent mode. Conclusions H2S can increased the expression KATP channel SUR2B mRNA and Kir6.1 mRNA levels of ASMC.