Bound thrombin-induced upregulation of myosin heavy chain isoform, SMemb messenger RNA expression in cultured rabbit vascular smooth muscle cells.
Bound thrombin-induced upregulation of myosin heavy chain isoform, SMemb messenger RNA expression in cultured rabbit vascular smooth muscle cells.
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培养的兔血管平滑肌细胞中结合凝血酶诱导的肌球蛋白重链亚型、SMemb 信使 RNA 表达上调。
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发表时间：
2003
期刊：
影响因子：
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通讯作者：
T. Kosugi
中科院分区：
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作者：
S. Shimada;M. Sunagawa;M. Nakamura;T. Kosugi
为探讨凝血酶是否能诱导血管平滑肌细胞SMemb表达的调节作用，采用原位杂交（ISH）和逆转录-聚合酶链反应（RT-PCR）技术检测了体外培养的兔主动脉平滑肌细胞SMemb mRNA的表达。为了测试结合的凝血酶对SMemb表达的浓度和时间依赖性作用，将汇合的VSM细胞在含有0、3、10和30单位/ml结合的凝血酶的10% FBS-DMEM中孵育48小时。此外，将汇合的VSM细胞在含有10单位/ml结合凝血酶的10% FBS-DMEM中孵育6、12、24和48小时。因此，结合凝血酶显着增加SMemb mRNA的浓度和时间依赖性的方式。与兔纤维蛋白原（10 μ g/ml）和天然凝血酶（10单位/ml）的作用相比，结合凝血酶显著增加SMemb mRNA，天然凝血酶略有增加，但纤维蛋白原不增加。其他肌球蛋白重链（MHC）亚型（SM 1和SM 2）的mRNA表达没有改变纤维蛋白原，天然凝血酶或绑定凝血酶。原位杂交结果显示，纤维蛋白原、天然凝血酶和结合凝血酶之间的MHC mRNA表达无显著差异。Western blot分析表明，SMemb蛋白水平显着增加了2.5倍，结合凝血酶。当在含有天然凝血酶或结合凝血酶的培养基中测定从0.5至48小时的凝块形成活性时，结合凝血酶的活性比天然凝血酶的活性下降得更慢。总之，结合凝血酶可上调SMemb mRNA和蛋白在培养的VSM细胞中的表达，并且结合凝血酶的活性在培养基中比天然凝血酶维持更长的时间。
To investigate whether bound thrombin can induce modulation of SMemb expression in vascular smooth muscle (VSM) cells, messenger RNA (mRNA) expression was measured by in situ hybridization (ISH) and reverse transcription-polymerase chain reaction (RT-PCR) in cultured rabbit aortic VSM cells. To test the concentration- and time-dependent effect of bound thrombin on the expression of SMemb, confluent VSM cells were incubated for 48 h in 10% FBS-DMEM containing 0, 3, 10 and 30 units/ml of bound thrombin. In addition, the confluent VSM cells were incubated for 6, 12, 24 and 48 h in 10% FBS-DMEM containing 10 units/ml of bound thrombin. Consequently, bound thrombin significantly increased SMemb mRNA in a concentration- and time-dependent manner. When compared with the effect of rabbit fibrinogen (10 microg/ml) and native thrombin (10 units/ml), SMemb mRNA was significantly increased by bound thrombin and was slightly increased by native thrombin, but not by fibrinogen. Other myosin heavy chain (MHC) isoform (SM1 and SM2) mRNA expressions were not changed by fibrinogen, native thrombin or bound thrombin. ISH revealed that there was no significant difference in the expression of MHC mRNAs among fibrinogen, native thrombin or bound thrombin. Western blot analysis demonstrated that the SMemb protein level was significantly increased by 2.5-fold by bound thrombin. When the clot-forming activities in cultured medium containing native thrombin or bound thrombin were measured from 0.5 to 48 h, the activity of bound thrombin declined more slowly than that of native thrombin. In conclusion, bound thrombin could upregulate the expression of SMemb mRNA and protein in cultured VSM cells and the activity of bound thrombin was maintained for longer than that of native thrombin in culture medium.