肝星状细胞合成气体信号分子H2S的测定及其意义

Endogenous production of gaseous signal molecule hydrogen sulphide by activated hepatic stellate cells

目的:测定活化肝星状细胞(hepaticstellatecells,HSC)H2S的生成率及对细胞增殖的影响.方法:采用活化大鼠HSC-T6,分4组:对照组、炔丙基甘氨酸(D-Propargylglycine,DL-PPG)组、N-硝基-L-精氨酸甲酯(Nitro-L-argininemethylester,L-NAME)组、氯化高铁血红素(hemin)组,每组重复6个皿.RT-PCR检测HSC-T6胱硫醚-γ-裂解酶(cystathionine-γ-lyase,CSE)mRNA水平,亚甲基蓝分光光度法测定H2S生成率,MTT法观察不同浓度外源性H2S供体-NaSH(0,50,100,500,1000μmol/L)对HSC-T6增殖的影响.结果:HSC-T6能自分泌H2S,DL-PPG可明显减少HSC-T6H2S生成率(4.55nmol/min±1.06nmol/minvs6.79nmol/min±1.27nmol/min,P<0.05).RT-PCR显示:HSC-T6CSEmRNA阳性,DL-PPG、hemin均降低HSC-T6CSEmRNA水平(P<0.05);L-NAME对HSC-T6CSEmRNA无明显影响.50μmol/LNaSH可明显促进HSC-T6增殖(细胞存活率为116%),500-1000μmol/LNaSH对细胞增殖无明显影响.结论:活化HSC自分泌H2S,并且影响其增殖.H2S在肝纤维化及肝硬化门脉高压症的发生机制可能有重要作用.

AIM： To assess whether hydrogen sulphide （H2S） is produced by activated hepatic stellate cells （HSC） and to investigate the effects of exogenous H2S on HSC proliferation. METHODS： HSC-T6, an activated rat HSC line, was used in the study. HSC-T6 cells were divided into four groups： control group, DL-PPG [Dpropargylglycine, an inhibitor of cystathionine γ-lyase （CSE）] group, L-NAME （nitro-L-arginine methyl ester） group, and hemin [a stimulator of heme oxygenase-1 （HO-1）] group. HSC-T6 cell suspension （1×105 cells） was prepared with DMEM containing 10% fetal calf serum. The production of H2S was detected by methylene blue spectrophotometry. The expression of CSE mRNA was tested by reverse transcription-polymerase chain reaction （RT-PCR）. Subsequently, the effects of different concentrations of sodium hydrosulfide （NaSH; 0, 50, 100, 500 and 1000 μmol/L）, an exogenous H2S ＂donor＂, on HSC-T6 cell proliferation were observed by MTT assay. RESULTS： The rate of production of endogenous H2S by HSC-T6 cells was 6.79 nmol/min ± 1.27 nmol/min. DL-PPG significantly inhibited H2S production （4.55 nmol/min ± 1.06 nmol/ min, P〈0.05）. CSE mRNA was positively expressed in HSC-T6 cells as revealed by RT-PCR. CSE mRNA expression was down-regulated in HSC-T6 cells treated with DL-PPG or hemin （both P〈0.05）. NaSH at a concentration of 50 μmol/L significantly promoted cell proliferation. In contrast, NaSH at concentrations of 500-1000 μmol/L had no significant impact on cell proliferation. CONCLUSION： Activated HSC can produce H2S. CSE may play an important role in the synthesis of H2S by HSC. Endogenous H2S produced by HSC may be related to the pathogenesis of cirrhotic portal hypertension.