奥曲肽对大鼠肝星状细胞胞质内游离钙及细胞增殖的影响

Effect of octreotide on regulation of intracellular free Ca^(2+) concentration of hepatic stellate cells in rats

目的:探讨在常氧和低氧条件下奥曲肽(octreotide)对大鼠肝星状细胞(HSC)胞内游离钙[Ca2+]i及增殖的调节.方法:采用钙荧光探针(Fura-2/AM)负载培养的大鼠HSC,观察常氧和低氧条件下培养48h后octreotide对HSC[Ca2+]i的调节,同时用四唑盐(MTT)比色法比较不同浓度的octreotide对大鼠HSC增殖的影响.环磷酸腺苷(cAMP)和环磷酸鸟苷(cGMP)放免分析药盒测cAMP,cGMP浓度.结果:与常氧条件相比,低氧条件下HSC[Ca2+]i显著升高(293.2±12.4nmol/Lvs137.7±7.8nmol/L.P<0.01).500、800和1000μg/Loctreotide在常氧状态下可引起HSCs[Ca2+]i降低(P<0.05),其值分别为92.5±2.5、83.8±2.3和76.6±2.2nmol/L.低氧时500,800和1000gg/Loctreotide可引起HSCs[Ca2+],降低(P<0.01),其值分别为204.3±7.4、174.1±4.8和156.6±6.6nmol/L.常氧对照组A值(0.232±0.016)明显低于低氧对照组(0.533±0.036)(P<0.01);经500、800和1000μg/Loctreotide处理后,无论是常氧还是低氧状态下A值均明显降低(P<0.01).低氧条件下cAMP和cGMP含量不发生改变(P>0.05);无论是常氧还是低氧状态,经过octreotide500μg/L处理后,cAMP和cGMP含量与相应对照组相比增(cAMP:1.69±0.18pmol/mgvs1.10±0.32pmol/mg.1.87±0.30pmol/mgvs1.37±0.25pmol/mg,P<0.05;cGMP:1.08±0.24pmol/mgvs0.86±0.12pmol/mg.1.17±0.53pmol/mgvs0.89±0.20pmol/mg,P<0.05),而Octreotide800,1000μg/L组更高(cAMP:1.99±0.27,2.48±0.37pmol/mgvs1.10±0.32pmol/mg,P<0.01:2.09±0.35.2.24±0.15pmol/mgvs1.37±0.25pmol/mg,P<0.01:cGMp:1.24±0.17,1.31±0.29pmol/mgvs0.86±0.12pmol/mg,P<0.01;1.38±0.29,1.46±0.35pmol/mgvs0.89±0.20pmol/mg,P<0.01).结论:缺氧可通过第二信使系统促进HSC的增生,而Octreotide无论常氧还是低氧条件下剂量依赖性抑制HSCs增殖;cAMP,cGMP参与了对HSC的调节.

AIM： To investigate the effect of octreotide on the regulation of intracellular free Ca^2＋ concentration （[Ca^2＋]i） and proliferation of hepatic stellate cells （HSCs） in rats. METHODS： Fluorescence Ca^2＋ indicator Fura-2/AM was used to observe the [Ca^2＋]i of HSCs in normoxic and chronic hypoxic condition. The effects of octreotide on the proliferation of HSCs were assessed by MTT assay, and the levels of cyclic adenosine monophosphate （cAMP） and cyclic guanosine monophosphate （cGMP） were detected by radioimmunoassay. RESULTS： The [Ca^2＋]i in hypoxic condition was markedly increased as compared with that in normoxic condition （293.2 ± 12.4 nmol/L vs 137.7± 7.8 nmol/L, P〈0.01 ）. In normoxic condition, the level of [Ca^2＋]i decreased sharply after 500, 800 and 1 000μg/L octreotide treatment （92.52± 2.52, 83.77±2.30 and 76.58±2.21 nmol/L, respectively, P〈0.01）; In hypoxic condition, 500, 800 and 1 000 Bg/L octreotide caused significant reduction in [Ca^2＋]i（204.28± 7.41,174.08±4.77 and 156.75±6.59 nmol/L, respectively, P〈0.01）. MTT assay showed that 500, 800 and 1 000 μg/L octreotide reduced the value of optical density （A value） in normoxic （0.173±0.010, 0.138±0.009, 0.100±0.010,respectively） and hypoxic （0.443±0.027, 0.320±0.014, 0.230± 0.014, respectively） condition. After exposure to hypoxic condition, the level of cAMP was not significantly different from that of cGMP （P〉0.05）. The contents of cAMP and cGMP markedly increased after 500, 800, and 1 000 Bg/L octreotide treatment in norrnoxic （cAMP： 1.69± 0.18, 1.99 ± 0.27, 2.48 ± 0.37 pmol/mg vs 1.10 ± 0.32 pmol/mg, P〈0.05 or P〈0.01; cGMP： 1.08 ± 0.24, 1.24 ± 0.17, 1.31 ± 0.29 pmol/mg vs 0.86 ± 0.12 pmol/mg, P〈0.05 or P〈0.01） and hypoxic （cAMP： 1.87 ± 0.30, 2.09 ± 0.35, 2.24 ± 0.15 pmol/mg vs 1.37 ± 0.25 pmol/mg, P〈0.05 or P〈0.01; cGMP： 1.17 ± 0.53, 1.38 ± 0.29, 1.46 ± 0.35 pmol/mg vs 0.89 ± 0.20 pmol/mg, P〈0.05 or P〈0.01 ） condition as compared with those in the corresponding control groups. CONCLUSION： Hypoxia can promote the proliferation of HSCs through the second messenger system, while octreotide antagonizes this action in a dose-dependant manner in both hypoxic and normoxic conditions, cAMP and cGMP play certain roles in the regulation of HSCs.