| 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.
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