pp60～（c-src）及Jak2在凝血酶诱血管平滑肌细胞增生中的作用
摘要
目的 凝血酶参与血管平滑肌细胞(VSMCs)异常增生，但其增生与时相、浓度的关系，及pp60c-src、Jak2在其增生信号传递过程中的作用不清楚。本研究旨在对上述问题进行探讨。 方法 3-10代的VSMCs的单细胞悬液用于实验。1. 为明确凝血酶刺激24小时促VSMCs增殖的量效关系, 实验分六组：凝血酶组（0，0.1，0.5，1，3，10U/ml）。 2.为明确凝血酶促VSMCs增殖的时效关系（0，1，6，12，24，48，72小时）,实验分对照组(DMEM)、凝血酶（0.5U/ml和1.0U/ml）。 3、为明确pp60c-src及Jak2活性在凝血酶促VSMCs增殖中的作用,实验分对照组(DMEM)、凝血酶组（0.5U/ml）、凝血酶和PP1组（PP1，Src特异性抑制剂，TH0.5U/ml＋PP1 10μmol/L）、凝血酶和AG490组（AG490，Jak2特异性抑制剂，TH0.5U/ml＋AG490 50μmol/L）。4、用MTS法或β-N-乙酰氨基己糖苷酶活性法测凝血酶促VSMCs细胞增殖。5、用免疫沉淀分别测定pp60c-src及Jak2的活性、碱性成纤维细胞生长因子（bFGF）及血小板源生长因子-A链（PDGF—A）的含量 变化。结果 1、不同浓度的凝血酶对VSMCs有明显促增殖作用，但凝血酶浓度为0.1-10U/ml在24小时时对VSMCs的促增殖作用无显著差异。2、0.5(U/ml)和1.0(U/ml)两种浓度的凝血酶促VSMCs增殖相对于对照组，在各时间段均有明显促增殖作用，且二者作用程度相似，增殖曲线呈双峰样改变，增殖峰在1小时 (0.870±0.042和0.890±0.052 比 0.734±0.015 ,P<0.05)和24小时(1.049±0.112和1.040±0.130比0.706±0.041, P <0.05)。 3、凝血酶刺激组pp60 c-src活性在1小时（1.316±0.036比1.0±0.051，P <0.05）及12小时（1.523±0.036比1.0±0.051, P <0.05）均明显上升。相对凝血酶组，PP1有效的降低了凝血酶诱导VSMCs 1小时峰值 (0.723±0.014比 0.786±0.022, P <0.05 )及24小时的峰值(0.874±0.032比 0.982±0.042, P <0.05). 4 凝血酶组，PDGF自分泌高峰在6小时(1.51±0.051比1.0±0.043, P <0.01) 及12小时(1.46±0.029比1.0±0.043, P < 0.01)，bFGF自分泌高峰在1小时（2.1±0.081比1.0±0.056, P <0.01）。用PP1干预凝血酶诱VSMCs增殖组，与凝血酶组比较，PDGF在6小时(1.12±0.046比 1.51±0.051, P <0.01)、及12小时(0.75±0.039比 1.46±0.029, P <0.01)自分泌高峰、bFGF在1小时
（1.39±0.052比2.1±0.081, P <0.01）自分泌高峰均明显被抑制。5凝血酶刺激组Jak2的活性在6小时(1.231±0.041vs1.0±0.046, P <0.05)及12小时(1.295±0.047vs1.0±0.046, P <0.05)均明显上升。相对于凝血酶组，AG490有效的降低了凝血酶诱导VSMCs 1小时峰值 (0.693±0.022vs 0.794±0.036, P <0.05 ) 及24小时的峰值（0.798±0.042vs 0.992±0.039, P <0.05）。结论 1、凝血酶浓度在0.1-10U/ml之间促VSMCs增殖无浓度依赖性，提示凝血酶受体有饱和性。 2、凝血酶促VSMCs的增殖在时间上呈双峰样改变，提示凝血酶不仅有早期快速促VSMCs的增殖作用，而且有后期促VSMCs的增殖作用。3、由于pp60c-src活性第二高峰（12小时）在凝血酶促VSMCs第二增殖峰值（24小时）之前，提示pp60 c-src活性后期的增加参与了凝血酶促VSMCs后期增殖。4、pp60 c-src参与了凝血酶促VSMCs自分泌PDGF和bFGF。5、由于Jak2活性高峰（6小时及12小时）在凝血酶促VSMCs第二增殖峰值（24小时）之前，提示Jak2活性后期的增加参与了凝血酶促VSMCs后期增殖。
Objective To study the effect of thrombin on cultured Vascular Smooth Muscle Cells (VSMCs) proliferation and the role of pp60c-src or Jak2 activity on thrombin-induced VSMCs proliferation. Methods: Growth-arrested VSMCs were used in experiment. 1. To study the effect of thrombin-induced cells proliferations in different concentration at 24h, following groups were included: thrombin (0, 0.1, 0.5, 1, 3, 10U/ml); To study the phase effect of thrombin-induced cells proliferations at 0, 1, 6, 12, 24, 48, 72h respectively; following groups were divided: control (DMEM), thrombin (0.5 U/ml or 1.0U/ml); To study the role of pp60c-src or Jak2 activity in thrombin-induced cells proliferations, four groups were included: control (DMSO), thrombin (0.5U/ml), thrombin (0.5U/ml) plus PP1 (10μmol/L), thrombin (0.5U/ml) plus AG490 (50μmol/L) respectively. The activity of pp60c-src or Jak2 in cells and the content of PDGF or bFGF in culture liquid were investigated by immunoprecipitation at 0, 1, 6, 12, 24, 48, 72h.2. Cell proliferation was quantified by MTS or hexosaminidase activity method. Results: 1. VSMCs proliferation in all groups treated with thrombin (0.1, 0.5, 1, 3, 10U/ml) at 24h was similar. 2. Either thrombin group (0.5 or 1.0U/ml) presents similar cell proliferation curves with two peaks, occurring at 1h(0.870±0.042 and 0.890±0.052vs 0.734±0.015, P<0.05) and at 24h(1.049±0.112 and 1.040±0.130vs0.706±0.041, P <0.05). 3. Compared with 0h, the activity of pp60c-src in thrombin group increased significantly at 1h（1.316±0.036 vs1.0±0.051，P <0.05）and at 12h（1.523±0.036vs1.0±0.051, P <0.05）.Compared with thrombin group, PP1 decreased the thrombin-induced cells proliferations at 1h(0.723±0.014vs 0.786±0.022, P<0.05) and at 24h(0.874±0.032vs 0.982±0.042, P <0.05). 4. The level of PDGF in thrombin group increased significantly at 6h(1.51±0.051vs1.0±0.043, P <0.01) and at 12h(1.46±0.029vs1.0±0.043, P <0.01). The level of bFGF in thrombin group increased significantly at 1h（2.1±0.081vs1.0±0.056 P <0.01）. Compared with thrombin group, PP1 decreased the level of PDGF at 6h(1.12±0.046vs 1.51±0.051, P <0.01) and at 12h(0.75±0.039vs 1.46±0.029, P <0.01) or the level of bFGF at 1h（1.39±0.052vs2.1±0.081, P <0.01）. 5. The activity of Jak2 in thrombin group increased significantly at 6h(1.231±0.041vs1.0±0.046, P<0.05) and at 12h(1.295±0.047vs1.0±0.046, P <0.05). Compared with thrombin group, AG490 decreased the thrombin-induced cells proliferations at 1h(0.693±0.022vs 0.794±0.036, P <0.05) and at 24h(0.798±0.042vs 0.992±
0.039, P <0.05). Conclusions: 1. The receptor of thrombin had receptor saturation, because the effects of thrombin stimulated proliferation of VSMCs in a dose-independent with thrombin concentration from 0.1U/ml to 10U/ml. 2. Cell proliferation induced by thrombin had two peaks at 1 hr and 24 hr. This indicated that induction of thrombin was not only quickly and transient but also torpid and late. 3. The increase of pp60c-src activity occurs before cell proliferation, which suggests that pp60c-src is involved in signal transmission pathway of thrombin-induced cell proliferation. 4. The pp60c-src activity is related to thrombin-induced autocrine of PDGF and bFGF. 5. The increase of Jak2 activity occurs before cell proliferation, which suggests that Jak2 is involved in signal transmission pathway of thrombin-induced cell proliferation.
（1.39±0.052比2.1±0.081, P <0.01）自分泌高峰均明显被抑制。5凝血酶刺激组Jak2的活性在6小时(1.231±0.041vs1.0±0.046, P <0.05)及12小时(1.295±0.047vs1.0±0.046, P <0.05)均明显上升。相对于凝血酶组，AG490有效的降低了凝血酶诱导VSMCs 1小时峰值 (0.693±0.022vs 0.794±0.036, P <0.05 ) 及24小时的峰值（0.798±0.042vs 0.992±0.039, P <0.05）。结论 1、凝血酶浓度在0.1-10U/ml之间促VSMCs增殖无浓度依赖性，提示凝血酶受体有饱和性。 2、凝血酶促VSMCs的增殖在时间上呈双峰样改变，提示凝血酶不仅有早期快速促VSMCs的增殖作用，而且有后期促VSMCs的增殖作用。3、由于pp60c-src活性第二高峰（12小时）在凝血酶促VSMCs第二增殖峰值（24小时）之前，提示pp60 c-src活性后期的增加参与了凝血酶促VSMCs后期增殖。4、pp60 c-src参与了凝血酶促VSMCs自分泌PDGF和bFGF。5、由于Jak2活性高峰（6小时及12小时）在凝血酶促VSMCs第二增殖峰值（24小时）之前，提示Jak2活性后期的增加参与了凝血酶促VSMCs后期增殖。
Objective To study the effect of thrombin on cultured Vascular Smooth Muscle Cells (VSMCs) proliferation and the role of pp60c-src or Jak2 activity on thrombin-induced VSMCs proliferation. Methods: Growth-arrested VSMCs were used in experiment. 1. To study the effect of thrombin-induced cells proliferations in different concentration at 24h, following groups were included: thrombin (0, 0.1, 0.5, 1, 3, 10U/ml); To study the phase effect of thrombin-induced cells proliferations at 0, 1, 6, 12, 24, 48, 72h respectively; following groups were divided: control (DMEM), thrombin (0.5 U/ml or 1.0U/ml); To study the role of pp60c-src or Jak2 activity in thrombin-induced cells proliferations, four groups were included: control (DMSO), thrombin (0.5U/ml), thrombin (0.5U/ml) plus PP1 (10μmol/L), thrombin (0.5U/ml) plus AG490 (50μmol/L) respectively. The activity of pp60c-src or Jak2 in cells and the content of PDGF or bFGF in culture liquid were investigated by immunoprecipitation at 0, 1, 6, 12, 24, 48, 72h.2. Cell proliferation was quantified by MTS or hexosaminidase activity method. Results: 1. VSMCs proliferation in all groups treated with thrombin (0.1, 0.5, 1, 3, 10U/ml) at 24h was similar. 2. Either thrombin group (0.5 or 1.0U/ml) presents similar cell proliferation curves with two peaks, occurring at 1h(0.870±0.042 and 0.890±0.052vs 0.734±0.015, P<0.05) and at 24h(1.049±0.112 and 1.040±0.130vs0.706±0.041, P <0.05). 3. Compared with 0h, the activity of pp60c-src in thrombin group increased significantly at 1h（1.316±0.036 vs1.0±0.051，P <0.05）and at 12h（1.523±0.036vs1.0±0.051, P <0.05）.Compared with thrombin group, PP1 decreased the thrombin-induced cells proliferations at 1h(0.723±0.014vs 0.786±0.022, P<0.05) and at 24h(0.874±0.032vs 0.982±0.042, P <0.05). 4. The level of PDGF in thrombin group increased significantly at 6h(1.51±0.051vs1.0±0.043, P <0.01) and at 12h(1.46±0.029vs1.0±0.043, P <0.01). The level of bFGF in thrombin group increased significantly at 1h（2.1±0.081vs1.0±0.056 P <0.01）. Compared with thrombin group, PP1 decreased the level of PDGF at 6h(1.12±0.046vs 1.51±0.051, P <0.01) and at 12h(0.75±0.039vs 1.46±0.029, P <0.01) or the level of bFGF at 1h（1.39±0.052vs2.1±0.081, P <0.01）. 5. The activity of Jak2 in thrombin group increased significantly at 6h(1.231±0.041vs1.0±0.046, P<0.05) and at 12h(1.295±0.047vs1.0±0.046, P <0.05). Compared with thrombin group, AG490 decreased the thrombin-induced cells proliferations at 1h(0.693±0.022vs 0.794±0.036, P <0.05) and at 24h(0.798±0.042vs 0.992±
0.039, P <0.05). Conclusions: 1. The receptor of thrombin had receptor saturation, because the effects of thrombin stimulated proliferation of VSMCs in a dose-independent with thrombin concentration from 0.1U/ml to 10U/ml. 2. Cell proliferation induced by thrombin had two peaks at 1 hr and 24 hr. This indicated that induction of thrombin was not only quickly and transient but also torpid and late. 3. The increase of pp60c-src activity occurs before cell proliferation, which suggests that pp60c-src is involved in signal transmission pathway of thrombin-induced cell proliferation. 4. The pp60c-src activity is related to thrombin-induced autocrine of PDGF and bFGF. 5. The increase of Jak2 activity occurs before cell proliferation, which suggests that Jak2 is involved in signal transmission pathway of thrombin-induced cell proliferation.
引文
McNamara CA, Sarembock IJ, Gimple LW, et al.Thrombin stimulates proliferation of cultured rat aortic smooth muscle cells by a proteolytically activated receptor. J Clin Invest 1993; 91: 94-98
Graham DJ, Alexander JJ. The effects of thrombin on bovine aortic endothelial and smooth muscle cells. J Vasc Surg 1990; 11: 307-310
Herbert JM, Lamarche I, Dol F. Induction of vascular muscle cell growth by selective activation of the thrombin receptor: effect of heparin. FERS Lett 1992; 301: 155-159
Kanthou C, Parray G, Wijelath E, et al. Thrombin-induced proliferation and expression of platelet-derived growth factor A-chain gene in human vascular smooth muscle cell. FEBS Lett 1992; 314: 143-148
MeBride, W, R.A. Lange and L. D. Hillis. Restenosis after successful coronary angioplasty. N.Engl.J. Med. 1988, 318: 1734-1737
Chen LB ,Teng NNH, Buchaman JM . Mitogenicity of thrombin and surface alteration on mouse spenocyte. Exp Cell Res,1976;101:41-46
Weiss RH, Maduri M. The mitogenic of thrombin in vascular smooth muscle cells is largely due to basic fibroblast growth factor . J Biol Chem 1993; 268: 5724-5727
Grand RJ, Turnell M,Grabham PW.Cellular consequences of thrombin-receptor activation. Biochem J 1996;313:353-368
Viswanathan M, Seltzer A, Saavedra JM,et al. Heterogeneous expression of angiotens in II AT1 receptors in neointim a of rat carotid artery and aorta after balloon catheter injury. Peptides 1994; 15: 1205-1211
Della Rocca GJ, Van Biesen T,Daaka Y,et al . Rea-dependent mitogen-activated protein kinase activation by G protein-coupled receptors, J Biol Chem 1997; 272: 19125-19132
Bydlowski SP, Pares MM. Stimulation of human smooth muscle cell proliferation by thrombin involves increased synthesis of Platet-dierived growth factor [J]. Chest, 1998; 114: 236-240
Nelken NA, Soifer SJ, Okeefe J, et al. Thrombin receptor expression in normal and atherosclerotic human arteries. J Clin Invest 1992; 90: 1614-1621
Guinebault C, Payrastre B, Sultan C, et al. Tyrosine kinases and phosphoinositide metabolism in thrombin-stimulated human platelets. Biochem J 1993; 292: 851-856
Coughlin SR.Sol Sherry lecture in thrombosis: how thrombin “talk” to cell: molecular mechanisms and roles in volv. Arterioscler Thromb Vasc Biol 1998;18514-18518
Molloy CJ, Taylor DS, Weber H: Angiotensin II stimulation of rapid protein tyrosine phosphorylation and protein kinase activation in rat aortic smooth muscle cells. J Biol Chem 1993; 268: 7338-7345
Bachhuber BG, Sarembock IJ, Gimple LW, et al. Thrombin-induced mitogenesis in cultured aortic smooth muscle cells requires prolonged thrombin exposure. Am J Physiol 1995; 268: C1141-1147
Cucina A, Borrelli V, Di Carlo A, et al. Thrombin induces production of growth factors from aortic smooth muscle cells. J Surg Res 1999; 82: 61-69
张茜，姜玉新，刘东青, 等.反义凝血酶受体基因表达对猪主动脉平滑肌细胞增殖的抑制作用.中华医学杂志，1999；79: 365-368
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Fager G. Thrombin and proliferation of vascular smooth muscle cell. Circ Res 1995; 77: 645-650
Hirata Y, Takagi Y, Fukuda Y, et al. Endothelin is a potent mitogen for rat vascular smooth muscle cells. Atherosclerosis 1989; 78: 225-228
Jawien A, Bowen-Pope DF, Lindner V, et al. Platelet-derived growth factor promotes smooth muscle migration and intimal thickening in a rat model of balloon angioplasty. J Clin Invest 1992; 89(2): 507-
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Graham DJ, Alexander JJ. The effects of thrombin on bovine aortic endothelial and smooth muscle cells. J Vasc Surg 1990; 11: 307-310
Herbert JM, Lamarche I, Dol F. Induction of vascular muscle cell growth by selective activation of the thrombin receptor: effect of heparin. FERS Lett 1992; 301: 155-159
Kanthou C, Parray G, Wijelath E, et al. Thrombin-induced proliferation and expression of platelet-derived growth factor A-chain gene in human vascular smooth muscle cell. FEBS Lett 1992; 314: 143-148
MeBride, W, R.A. Lange and L. D. Hillis. Restenosis after successful coronary angioplasty. N.Engl.J. Med. 1988, 318: 1734-1737
Chen LB ,Teng NNH, Buchaman JM . Mitogenicity of thrombin and surface alteration on mouse spenocyte. Exp Cell Res,1976;101:41-46
Weiss RH, Maduri M. The mitogenic of thrombin in vascular smooth muscle cells is largely due to basic fibroblast growth factor . J Biol Chem 1993; 268: 5724-5727
Grand RJ, Turnell M,Grabham PW.Cellular consequences of thrombin-receptor activation. Biochem J 1996;313:353-368
Viswanathan M, Seltzer A, Saavedra JM,et al. Heterogeneous expression of angiotens in II AT1 receptors in neointim a of rat carotid artery and aorta after balloon catheter injury. Peptides 1994; 15: 1205-1211
Della Rocca GJ, Van Biesen T,Daaka Y,et al . Rea-dependent mitogen-activated protein kinase activation by G protein-coupled receptors, J Biol Chem 1997; 272: 19125-19132
Bydlowski SP, Pares MM. Stimulation of human smooth muscle cell proliferation by thrombin involves increased synthesis of Platet-dierived growth factor [J]. Chest, 1998; 114: 236-240
Nelken NA, Soifer SJ, Okeefe J, et al. Thrombin receptor expression in normal and atherosclerotic human arteries. J Clin Invest 1992; 90: 1614-1621
Guinebault C, Payrastre B, Sultan C, et al. Tyrosine kinases and phosphoinositide metabolism in thrombin-stimulated human platelets. Biochem J 1993; 292: 851-856
Coughlin SR.Sol Sherry lecture in thrombosis: how thrombin “talk” to cell: molecular mechanisms and roles in volv. Arterioscler Thromb Vasc Biol 1998;18514-18518
Molloy CJ, Taylor DS, Weber H: Angiotensin II stimulation of rapid protein tyrosine phosphorylation and protein kinase activation in rat aortic smooth muscle cells. J Biol Chem 1993; 268: 7338-7345
Bachhuber BG, Sarembock IJ, Gimple LW, et al. Thrombin-induced mitogenesis in cultured aortic smooth muscle cells requires prolonged thrombin exposure. Am J Physiol 1995; 268: C1141-1147
Cucina A, Borrelli V, Di Carlo A, et al. Thrombin induces production of growth factors from aortic smooth muscle cells. J Surg Res 1999; 82: 61-69
张茜，姜玉新，刘东青, 等.反义凝血酶受体基因表达对猪主动脉平滑肌细胞增殖的抑制作用.中华医学杂志，1999；79: 365-368
Du J, Brink M, Peng T, et al. Thrombin regulates insulin-like growth factor-1 receptor transcription in vascular smooth muscle: characterization of the signaling pathway. Circ Res 2001; 88: 1044-1055
Marrero MB, Schieffer B, Paxton WG ,et al. Electroporation of pp60c-src antibodies inhibits angiotensin II activation ofphospholipase C-γ1 in rat aortic smooth muscle cells. J Biol Chem 1995; 270: 15734-15738
Marrero MB, Schieffer B,Paxton WG, et al. Direct stimulation of Jak/ STAT pathway by
the Angiotensin II AT1 receptor . Nature 1995;375: 247-250
Marrero MB, Schieffer B,Li B,et al. Role of Janus kinase/signal tranducer and activiator of transcription and mitogen- activated protein kinase cascades in Angiotensin II-and Platelet-derived growth factor-induced vascular smooth muscle cell proliferation. J Biol Chem1997;272:24684-24690
Madamanchi NR ,Li S, Patterson C ,ea tl.Thrombin regulates vascular smooth cell growth and heat shock proteins via JAK-STAT pathway,Biochem J,2001;276:18915-18924
Ma H, Matsunaga H, Li B, et al. Regulation of PDGF-beta receptor-operated Ca2+ channels by phospholipase C-gamma 1 in glomerular mesangial cells. Am J Physiol 1996; 271: F994-F1003
Malarkey K, Belham CM, Paul A, et al. The regulation of tyrosine kinase signalling pathways by growth factor and G-protein-coupled receptors. Biochem J 1995; 309: 361- 375
Fager G. Thrombin and proliferation of vascular smooth muscle cell. Circ Res 1995; 77: 645-650
Hirata Y, Takagi Y, Fukuda Y, et al. Endothelin is a potent mitogen for rat vascular smooth muscle cells. Atherosclerosis 1989; 78: 225-228
Jawien A, Bowen-Pope DF, Lindner V, et al. Platelet-derived growth factor promotes smooth muscle migration and intimal thickening in a rat model of balloon angioplasty. J Clin Invest 1992; 89(2): 507-
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