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

摘要

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48 h 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±12.4 nmol/L vs 137.7±7.8 nmol/L, P<0.01).500、800和1 000 μg/L octreotide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±2.2 nmol/L.低氧时500,800和1 000 μg/L octreotide可引起HSCs[Ca²⁺]ｉ降低(P<0.01), 其值分别为204.3±7.4、174.1±4.8和156.6±6.6 nmol/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1 000 μg/L octreotide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octreotide 500 μg/L处理后, cAMP和cGMP含量与相应对照组相比增高(cAMP: 1.69±0.18 pmol/mg vs 1.10±0.32 pmol/mg, 1.87±0.30 pmol/mg vs 1.37±0.25 pmol/mg, P<0.05; cGMP: 1.08±0.24 pmol/mg vs 0.86±0.12 pmol/mg, 1.17±0.53 pmol/mg vs 0.89±0.20 pmol/mg, P<0.05), 而Octreotide 800, 1 000 μg/L组更高(cAMP: 1.99±0.27, 2.48±0.37 pmol/mg vs 1.10±0.32 pmol/mg, P<0.01; 2.09±0.35, 2.24±0.15 pmol/mg vs 1.37±0.25 pmol/mg, P<0.01; cGMP: 1.24±0.17, 1.31±0.29 pmol/mg vs 0.86±0.12 pmol/mg, P<0.01; 1.38±0.29, 1.46±0.35 pmol/mg vs 0.89±0.20 pmol/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.

关键词: 奥曲肽; 钙; 肝; 星状细胞; 大鼠; cAMP; cGMP

Effect of octreotide on regulation of intracellular free Ca²⁺ concentration of hepatic stellate cells in rats

Xiu-Lan Xue, Ju-Sheng Lin, Xue-Mei Sun, He-Jun Zhou

Xiu-Lan Xue, Ju-Sheng Lin, Xue-Mei Sun, He-Jun Zhou, Institute of Liver Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China

Correspondence to: Ju-Sheng Lin, Institute of Liver Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China. linjusheng2001@163.com

Received: June 6, 2005
Revised: June 10, 2005
Accepted: June 13, 2005
Published online: August 28, 2005

Abstract

AIM: To investigate the effect of octreotide on the regulation of intracellular free Ca²⁺ concentration ([Ca²⁺]i) and proliferation of hepatic stellate cells (HSCs) in rats.

METHODS: Fluorescence Ca²⁺ indicator Fura-2/AM was used to observe the [Ca²⁺]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 [Ca²⁺]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 [Ca²⁺]i decreased sharply after 500, 800 and 1 000 mg/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 mg/L octreotide caused significant reduction in [Ca²⁺]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 mg/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 μg/L octreotide treatment in normoxic (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.

Key Words: Octreotide; Ca²⁺; Liver; Hepatic stellate cell; Rats; Cyclic adenosine monophosphate; Cyclic guanosine monophosphate

0 引言

肝硬化主要的特征是假小叶的形成、血管结构发生紊乱和门静脉高压.许多研究表明肝星状细胞在肝纤维化中起着重要作用.而在这一过程中, Ca²⁺作为最重要的信使物质, 无疑起着举足轻重的作用[1].生长抑素类似物奥曲肽能抑制多种肿瘤细胞的生长[2].现研究奥曲肽对常氧和低氧状态下HSC[Ca²⁺]ｉ的调节及细胞增殖的影响, 旨在探讨其抗肝纤维化机制.

1 材料和方法

1.1 材料

大鼠的肝星状细胞由美国Scott L.Friedman教授惠赠, 其表型为活化的HSC.100 mL/L胎牛血清(GIBCO)DMEM.常氧(37℃, 50 mL/L CO₂, 210 mL/L O₂, 740 mL/L N₂)和低氧(37℃, 50 mL/L CO₂, 50 mL/L O₂, 900 mL/L N2)培养箱(常氧培养箱: FPRMA 311 CO₂ Incubator美国制造; 低氧培养箱: Galaxy R CO₂ Incubator, 英国制造).奥曲肽(octreotide北京诺华制药有限公司.Fura-2/AM(Sigma, 美国).

1.2 方法

HSC铺满瓶底(80-90)%后1∶2传代, 待第2代细胞长至60%左右融合时, 换无血清培养基24 h同步化后, 常氧(N)及低氧(H)组均分别分为4组, 用含有下列3种不同浓度的octreotide的无血清培养基处理: 对照组(NＣ, HＣ); 500 μg/L octreotide组(Noct1, Hoct1); 800 μg/L octreotide组(N_oct2, H_oct2); 1 000 μg/L octreotide组(N_oct3, H_oct3)组.每组HSC分别进行常氧和缺氧培养.

1.2.1 HSC的[Ｃa²⁺]ｉ测定: 取汇合成单层的HSCs, 弃培养基, Hanks液洗涤3次, 用含2 mmol/L EDTA的D-Hanks 37℃温育5 min, 轻柔吹打, 离心, Hanks液洗涤3次并制成细胞悬液.台盼蓝排斥实验检查, 细胞存活率在95%以上, 调整细胞密度为109/L, 将悬液分数管.向细胞悬液内加入终浓度为5 μmol/L Fura-2/AM(Sigma, 美国), 37℃恒温避光震荡30 min, 离心弃上清后将细胞重新悬浮于Hanks液中, 于2 h内测定.另备未负载Fura-2的细胞以测定自身荧光.采用RF-5301PC荧光分光光度计(Shimadiu, 日本)进行[Ca²⁺]ｉ的荧光测定, 激发波长340 nm, 380 nm, 发射波长480 nm, 分别加入Triton和EGTA测量最大和最小值.按照Grynkiewicz et al的方法计算, 每次平行检测8个样品.测定液用超纯水配制, 并在测定缺氧细胞时用缺氧气体预平衡.

1.2.2 MTT法检测细胞增殖: 收集对数生长期的细胞, 调整密度为1×10⁴/孔接种于96孔培养板, 继续培养24 h后加入500, 800和1 000 μg/L octreotide分别在常氧、低氧培养箱培养48 h吸出培养液, 加入0.5 g/L的MTT 10 μL作用4 h, 吸出培养液加入二甲基亚砜150 μL并混合.测吸光度值计算细胞存活率.选择细胞存活90%以上的作为试验主要药物浓度.每组平行检测8孔, 设1孔只加DMSO不加MTT为空白对照, 用酶标仪在570 nm处测各孔吸光度A值以反映活细胞数目.

1.2.3 cAMP, cGMP含量测定: 各组弃培养液, 立刻加入冰冷的0.24 mol/L高氯酸1 mL, 刮取收集细胞, 冰浴下超声破碎, 离心取上清以3 mol/L KOH中和至pH 6.3左右, 离心除去高氯酸钾沉淀, -20℃贮存备测.cAMP, cGMP测定按上海中医学院药盒说明进行.

统计学处理 所有结果均以均数±标准误表示, 多组间的比较采用F检验, 组间的两两比较采用ｑ检验.用Sigmaplα2000软件处理数据并作图.

2 结果

2.1 octreotide对HSC[Ca2+]ｉ的影响

低氧培养后HSC[Ca²⁺]ｉ显著高于常氧状态(P<0.01); 常氧状态下分别经过octreotide 500, 800和1 000 μg/L处理后, [Ca²⁺]ｉ降低(P<0.05), 而低氧培养后[Ca²⁺]ｉ明显降低(P<0.01)(表1).

表1 Octreotide对HSC[Ca²⁺]ｉ(nmol/L)的影响.

	Control	Octreotide 500 μg/L	Octreotide 800 μg/L	Octreotide 1 000 μg/L
Normoxia(Ｎ)	137.7±7.8	92.5±2.5 a	83.8±2.3a	76.6±2.2a
Hypoxia(Ｈ)	293.2±12.4b	204.3±7.4b	174.1±4.8b	156.8±6.6b

^aP<0.05,

^bP<0.01 vs 常氧对照组.

2.2 octreotide对HSCs增殖的影响

低氧对照组A值明显高于常氧对照组(P<0.01); 500, 800, 和1 000 μg/L octreotide处理组无论是常氧还是低氧状态下Ａ值均明显降低(P<0.01, 表2).

表2 Octreotide对HSC增殖的影响.

	Control	Oct1(500 μg/L)	Oct2(800 μg/L)	Oct3(1 000 μg/L)
Normoxia(Ｎ)	0.232±0.016	0.1 725±0.010a	0.138±0.009a	0.100±0.010a
Hypoxia(Ｈ)	0.533±0.036b	0.443±0.027b	0.320±0.014b	0.230±0.014b

^aP<0.05,

^bP<0.01 vs 常氧对照组.

2.3 缺氧对HSC cAMP, cGMP的含量的影响

缺氧时HSCs cAMP, cGMP含量不发生改变(P>0.05); 无论是常氧还是低氧状态下经octreotide 500 μg/L处理后, cAMP和cGMP含量增高(P<0.05), 而octreotide 800, 1 000 μg/L组更高(P<0.01, 表3, 4).

表3 Octreotide对HSC cAMP(pmol/mg)影响.

	Control	Octreotide 500 μg/L	Octreotide 800 μg/L	Octreotide 1 000 μg/L
Normoxia(Ｎ)	1.10±0.32	1.69±0.18a	1.99±0.27b	2.48±0.37b
Hypoxia(Ｈ)	1.37±0.25	1.87±0.30c	2.09±0.35d	2.24±0.15d

^aP<0.05,

^bP<0.01 vs 常氧对照组;

^cP<0.05,

^dP<0.01 vs 低氧对照组.

表4 Octreotide对HSC cGMP(pmol/mg)的影响.

	Control	Octreotide 500 μg/L	Octreotide 800 μg/L	Octreotide1 000 μg/L
Normoxia(Ｎ)	0.86±0.12	1.08±0.24a	1.24±0.17b	1.31±0.29b
Hypoxia(Ｈ)	0.89±0.20	1.17±0.53c	1.38±0.29d	1.46±0.35d

^aP<0.05,

^bP<0.01 vs 常氧对照组;

^cP<0.05,

^dP<0.01 vs 低氧对照组.

3 讨论

HSC的激活增殖是纤维化发生发展的中心环节[3].肝纤维化恢复期, 激活的HSC减少使胞外基质(ECM)分泌减少, 促进ECM的降解, 因此在肝纤维化中有重要作用.Ca²⁺在细胞增殖中起重要作用.Ca²⁺升高一方面激活胞质中的收缩蛋白, 另一方面激活丝裂原活化的蛋白激酶(mitogen-activated proteinkinase, MAPK)和胞核Ca²⁺迅速增加[4].这些因素共同作用的结果使静止期的细胞进入细胞周期, 从而引起细胞的增殖.缺氧可能通过激活HSC, 释放血小板源生长因子和内皮素、Ca²⁺等来直接促进HSC的增殖.

生长抑素类似物octreotide能抑制多种肿瘤细胞的生长[2]; 也可以引起HSC舒张, 从而降低降低门静脉血流[5]; 还助于抗纤维化[6], 而关于此方面的体外研究未见报道.因此我们采用体外培养HSC, 观察了octreotide在常氧, 低氧培养下[Ca²⁺]ｉ及细胞增殖的调节及其cAMP, cGMP的含量变化.结果提示octreotide在HSC激活、增殖这一致纤维化的中心环节中起重要作用, 这种作用主要通过HSC内[Ca²⁺]ｉ, cAMP和cGMP实现的.可能是octreotide可以激活可溶性鸟苷酸环化酶(soluble guanglyl cyclase, SGC), 调节cGMP的合成, 而cGMP在许多组织特别是平滑肌中起着重要的调节作用.实验证明, 外源性octreotide既可以通过调节HSC内的cAMP和cGMP水平, 也可以通过调节细胞内[Ca²⁺]ｉ水平来发挥作用, 因此使HSC增殖受到抑制.结果表明octreotide在HSC激活、增殖这一致纤维化的中心环节中起重要作用, octreotide可能成为防治肝纤维化的重要途径.同时也应该注意到octreotide抗纤维化内在机制是多方面的, 也是非常复杂的, 其具体的机制尚待深入研究.

总之, 生长抑素类似物octreotide在常氧、低氧状态下对HSC cAMP, cGMP和[Ca²⁺]ｉ的调节及其增殖的影响, 为octreotide在肝硬化的治疗提供新的靶点.

备注

编辑: 潘伯荣 审读: 张海宁

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