【作者】 方正旭；

【导师】 程晓曙；

【作者基本信息】 江西医学院 ， 内科学， 2003， 硕士

【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<WP=4>（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。更多还原

【Abstract】 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±<WP=6>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.更多还原