硫化氢对体外大鼠肝星状细胞内Ca2+浓度变化及细胞增殖的影响

目的 研究硫化氢(H₂ S)对大鼠肝星状细胞-T6(HSC-T6) Ca²⁺浓度、细胞增殖的影响及其机制。 方法 活化HSC-T6用含10%小牛血清DMEM培养液制备为1×10⁵个肝星状细胞(HSC)悬液。钙离子荧光探针Fluo-3/AM负载细胞后，在不同刺激条件下，利用激光扫描共焦显微镜动态扫描HSC-T6细胞内Ca²⁺荧光强度（FI）变化，FI表示细胞内Ca²⁺浓度。四唑盐比色法，观察不同浓度H₂S供体——NaSH对HSC-T6细胞增殖的影响。 结果 低浓度H₂S（100μmol/L）明显降低HSC-T6细胞内Ca²⁺浓度（P<0.05)，而细胞增殖增加（增殖率为116%）；K_ATP通道阻断剂——格列本脲可阻断H2S的作用。高浓度H₂S（1mmol/L）刺激HSC-T6细胞内Ca₂₊浓度增加，但细胞增殖无明显变化(P＞0.05)。 结论 低浓度H₂S通过激活HSC-T6细胞K_ATP通道降低细胞内Ca²⁺浓度，可能通过调节细胞氧化应激促进细胞增殖；高浓度H2S刺激HSC-T6细胞内Ca₂₊浓度增加。提示H₂S在肝硬化门脉高压症的发生机制中具有双重作用。

EFFECTS OF HYDROGEN SULFIDE ON INTRACELLULAR CALCIUM AND PROLIFERATION IN HEPATIC STELLATE CELLS EM>IN VITRO/EM>

Objective To study the effect of hydrogen sulfide (HSUB>2/SUB>S) on intracellular calcium concentration and proliferation in hepatic stellate cells (HSC) and the possible mechanism of HSUB>2/SUB>S. Methods HSC-T6, an activated rat HSC, was plated in 35-mm culture dish at a density of 1×10SUP>5/SUP>/ml, and rotuinely incubated in DMEM media for 24 hours. The intracellular CaSUP>2+/SUP> loaded by Flu-3/AM was measured by laser scanning confocal microscope. The dynamic changes of intracellular CaSUP>2+/SUP> fluorescence intensity(［CaSUP>2+/SUP>］i ) stimulated by NaSH, a HSUB>2/SUB>S donor, were measured. The HSC proliferations were measured by MTT at different HSUB>2/SUB>S concentrations. Results Low concentration of HSUB>2/SUB>S(100μmol/L) decreased significantly HSC-T6 ［CaSUP>2+/SUP>］i (16.20±3.56 vs 14.12±3.76,EM>P/EM><0.05). The increased HSC-T6 proliferations were also observed in low concentration stimulation of HSUB>2/SUB>S(50-100μmol/L). The decreased ［CaSUP>2+/SUP>］i was inhibited by glibenclamide, a KSUB>ATP/SUB> channel antagonist， significantly when administered with HSUB>2/SUB>S of higher concentrations. However, high concentration of HSUB>2/SUB>S(1mmol/L) increased significantly HSC-T6 ［CaSUP>2+/SUP>］i，P<0.05, with no significant changes in proliferation. Conclusion Th