小G蛋白及MAPK在脂蛋白（a）促血管平滑肌细胞迁移中作用的研究
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摘要
第一部分脂蛋白(a)诱导的细胞骨架构建及其促血管平滑肌细胞迁移
目的：研究脂蛋白(a)所诱导的人血管平滑肌细胞的迁移及细胞骨架构建变化。方法：本研究用不同浓度的脂蛋白(a)[Lp(a)]诱导原代培养的人血管平滑肌细胞(VSMC)，通过迁移试验观察VSMC迁移数量的变化。Lp(a)诱导VSMC，在不同时间点用免疫荧光标记与细胞运动有关的肌动蛋白细胞骨架结构，激光共聚焦显微镜观察人VSMC细胞骨架构建变化情况。结果：Lp(a)的终浓度从40μg／ml至640μg／ml，其诱导的迁移细胞数随Lp(a)的终浓度的升高而增加(P＜0.05)。Lp(a)诱导VSMC10分钟后，激光共聚焦显微镜观察，可见细胞内出现张力纤维和粘着斑，并可见丝状伪足；20分钟后张力纤维和粘着斑明显增多；30分钟后细胞内的张力纤维和粘着斑较20分钟时无明显变化。结论：Lp(a)诱导后VSMC细胞骨架构建出现规律性的变化，从而导致VSMC的迁移。
第二部分RhoA及Rac1介导的脂蛋白(a)促血管平滑肌细胞迁移作用
目的：研究RhoA及Rac1在Lp(a)诱导的VSMC细胞骨架构建及迁移中的作用。方法：用脂质体将Rac1siRNA转移至VSMC内(RNAi)，阻断Rac1的表达，再用Lp(a)诱导阻断后的细胞，RT-PCR和Western blot检测RNA干扰效果，激光共聚焦显微镜观察细胞骨架构建变化，迁移试验观察VSMC迁移数量的变化。用脂质体将C3转移酶转移入VSMC阻断RhoA通路，再用Lp(a)诱导阻断后的细胞，激光共聚焦显微镜观察细胞骨架构建情况，迁移试验观察VSMC迁移数量的变化。结果：RT-PCR和Western blot检测均显示Rac1siRNA干扰效果明显，较未干扰组和对照siRNA组有显著差异(P＜0.01)。Rac1siRNA干扰人VSMC，Lp(a)诱导20分钟后，激光共聚焦显微镜观察，细胞内未见明显的张力纤维、粘着斑及丝状伪足。未干扰组VSMC迁移细胞数为77.47±5.53个，对照siRNA组为77.20±7.46个，Rac1siRNA组为40.13±4.84个，Rac1siRNA组迁移细胞数较未干扰和对照siRNA组均明显减少(P＜0.01)。C3转移酶阻断RhoA通路，Lp(a)诱导20分钟后，细胞内未见明显的张力纤维、粘着斑及丝状伪足。C3转移酶组VSMC迁移细胞数为42.47±6.06个，对照组为76.47±6.28个，C3转移酶组迁移细胞数较对照组明显减少(P＜0.01)。结论：Lp(a)诱导的VSMC细胞骨架构建及细胞迁移依赖于Rac1及RhoA的生物学活性。
第三部分ERK及p38在脂蛋白(a)促血管平滑肌细胞迁移中的作用
目的：研究ERK及p38在Lp(a)诱导的VSMC细胞骨架构建及迁移中的作用。方法：分别用不同浓度的ERK激酶抑制剂PD98059及p38激酶抑制剂SB202190抑制人VSMC内ERK及p38的活化，再用Lp(a)诱导处理后的细胞，Western blot检测P-ERK及P-p38的表达，激光共聚焦显微镜观察细胞骨架构建变化，迁移试验观察VSMC迁移数量的变化。结果：Western blot检测显示PD98059的终浓度在10μmol／L时P-ERK表达无明显变化(P＞0.05)，从20μmol／L至40μmol／L，随其终浓度的逐渐升高，P-ERK的表达逐渐降低(P＜0.01)，达到40μmol／L后，其抑制作用达到高峰；SB202190的终浓度从5μmol／L至20μmol／L，随其终浓度的逐渐升高，P-p38的表达逐渐降低(P＜0.01)，达到20μmol／L后，其抑制作用达到高峰。PD98059及SB202190分别预处理人VSMC，Lp(a)诱导20分钟后，细胞内未见明显的张力纤维、粘着斑及丝状伪足。对照组VSMC迁移细胞数为78.87±7.43个，PD98059处理组为49.20±6.47个，SB202190处理组为51.73±8.25个，PD98059处理组及SB202190处理组迁移细胞数均较正常对照组明显减少(P＜0.01)。结论：Lp(a)诱导的VSMC细胞骨架构建及细胞迁移依赖于ERK及p38的活化。
Part one Cytoskeleton reorganization and cell migration in human vascular smooth muscle cell stimulated by Lp(a)
Objectives: To study the reorganization of cytoskeleton and cell migration in human vascular smooth muscle cell (VSMC) stimulated by lipoprotein(a)[Lp(a)]. Methods: Firstly, the primary human VSMCs were cultured. Migration assays were applied to VSMC stimulated by Lp(a) of different concentrations. After VSMCs had been stimulated by Lp(a), F-actin was stained by FITC-labeled phalloidin to visualize actin-based structure including filopodia and stress fiber at different time points. And vinculin distribution of focal adhesion was visualized through indirect immunofluorescence at different time points too. These cytoskeletons of VSMC were then observed with confocal laser scanning microscope. Results: When the final concentration of Lp(a) was confined from 40μg/ml to 640μg/ml, the number of migrating VSMCs added while the final concentration was increased with significant difference (P<0.05) . The filopodia, stress fibers and focal adhesions in VSMC appeared at 10min after stimulation of Lp(a). The stress fibers and focal adhesions appeared remarkably at 20min. There was no evident change of these structures at 30min. Conclusion: Lp(a) induced the reorganization of cytoskeleton in human VSMC and stimulated cell migration of VSMC.
Part two The role of Rac1 and RhoA in cell migration of human VSMCs stimulated by Lp(a)
Objectives: To verify the effect of Rac1 and RhoA in cytoskeleton reorganization and cell migration of human VSMCs stimulated by Lp(a). Methods: The siRNA was introduced into cultured human VSMCs by lipofectamine to block the expression of
Rac1 gene. Then VSMCs were stimulated by Lp(a). Reverse-transcription polymerase chain reaction (RT-PCR) and western blot were applied to detect the efficacy of RNA interference. The cytoskeletons of these VSMCs were then observed with confocal laser scanning microscope. The number of migrated cells was determined by migration assays. C3 exoenzyme was introduced into VSMCs by lipofectamine to block the activity of RhoA. Then VSMCs were stimulated by Lp(a). The cytoskeletons of these VSMCs were then observed with confocal laser scanning microscope and the number of cells that had migrated was determined by migration assays. Results: The Rac1 siRNA demonstrated the efficiency in the blocking of Rac1, with significant difference from the control (P<0.01) . After VSMCs were stimulated by Lp(a) for 20min, there was no filopodia, stress fibers and focal adhesions in VSMC which Rac1 siRNA had been introduced into. The number of VSMCs that had migrated in the RNAi group was fewer than the control groups with significant difference (P<0.01) . After VSMCs were stimulated by Lp(a) for 20min, there was no filopodia, stress fibers and focal adhesions in VSMC which C3 exoenzyme had been introduced into. The number of VSMCs that had migrated in the C3 exoenzyme group was fewer than the control groups with significant difference (P<0.01) . Conclusion: Stimulation of VSMC cytoskeleton reorganization and migration by Lp(a) is dependent on Rac1 and RhoA.
Part three The role of ERK and p38 in cell migration of human VSMCs stimulated by Lp(a)
Objectives: To verify the effect of ERK and p38 in cytoskeleton reorganization and cell migration of human VSMCs stimulated by Lp(a). Methods: The ERK kinase inhibitor PD98059 of different concentrations and p38 kinase inhibitor SB202190 of different concentrations were applied to inhibit activation of ERK and p38 in human VSMCs. Then VSMCs were stimulated by Lp(a). Western blot were applied to detect the expression of P-ERK and P-p38. The cytoskeletons of these VSMCs were then observed with confocal laser scanning microscope. The number of cells that had migrated was determined by migration assays. Results: The expression of P-ERK was
decreasing while the final concentration of PD98059 was increased with significant difference (P<0.01) , when the final concentration of PD98059 was confined from 20μmol/L to 40μmol/L. The expression of P-p38 was decreasing while the final concentration of SB202190 was increased with significant difference (P<0.01) ,when the final concentration of SB202190 was confined from 5μmol/L to 20μmol/L. After VSMCs were stimulated by Lp(a) for 20min, there was no filopodia, stress fibers and focal adhesions in VSMCs which had been treated with PD98059 or SB202190. The number of VSMCs that had migrated in the PD98059 group and SB202190 group was fewer than the control groups with significant difference (P<0.01) . Conclusion: Stimulation of VSMC cytoskeleton reorganization and migration by Lp(a) is dependent on activation of ERK and p38.
目的：研究脂蛋白(a)所诱导的人血管平滑肌细胞的迁移及细胞骨架构建变化。方法：本研究用不同浓度的脂蛋白(a)[Lp(a)]诱导原代培养的人血管平滑肌细胞(VSMC)，通过迁移试验观察VSMC迁移数量的变化。Lp(a)诱导VSMC，在不同时间点用免疫荧光标记与细胞运动有关的肌动蛋白细胞骨架结构，激光共聚焦显微镜观察人VSMC细胞骨架构建变化情况。结果：Lp(a)的终浓度从40μg／ml至640μg／ml，其诱导的迁移细胞数随Lp(a)的终浓度的升高而增加(P＜0.05)。Lp(a)诱导VSMC10分钟后，激光共聚焦显微镜观察，可见细胞内出现张力纤维和粘着斑，并可见丝状伪足；20分钟后张力纤维和粘着斑明显增多；30分钟后细胞内的张力纤维和粘着斑较20分钟时无明显变化。结论：Lp(a)诱导后VSMC细胞骨架构建出现规律性的变化，从而导致VSMC的迁移。
第二部分RhoA及Rac1介导的脂蛋白(a)促血管平滑肌细胞迁移作用
目的：研究RhoA及Rac1在Lp(a)诱导的VSMC细胞骨架构建及迁移中的作用。方法：用脂质体将Rac1siRNA转移至VSMC内(RNAi)，阻断Rac1的表达，再用Lp(a)诱导阻断后的细胞，RT-PCR和Western blot检测RNA干扰效果，激光共聚焦显微镜观察细胞骨架构建变化，迁移试验观察VSMC迁移数量的变化。用脂质体将C3转移酶转移入VSMC阻断RhoA通路，再用Lp(a)诱导阻断后的细胞，激光共聚焦显微镜观察细胞骨架构建情况，迁移试验观察VSMC迁移数量的变化。结果：RT-PCR和Western blot检测均显示Rac1siRNA干扰效果明显，较未干扰组和对照siRNA组有显著差异(P＜0.01)。Rac1siRNA干扰人VSMC，Lp(a)诱导20分钟后，激光共聚焦显微镜观察，细胞内未见明显的张力纤维、粘着斑及丝状伪足。未干扰组VSMC迁移细胞数为77.47±5.53个，对照siRNA组为77.20±7.46个，Rac1siRNA组为40.13±4.84个，Rac1siRNA组迁移细胞数较未干扰和对照siRNA组均明显减少(P＜0.01)。C3转移酶阻断RhoA通路，Lp(a)诱导20分钟后，细胞内未见明显的张力纤维、粘着斑及丝状伪足。C3转移酶组VSMC迁移细胞数为42.47±6.06个，对照组为76.47±6.28个，C3转移酶组迁移细胞数较对照组明显减少(P＜0.01)。结论：Lp(a)诱导的VSMC细胞骨架构建及细胞迁移依赖于Rac1及RhoA的生物学活性。
第三部分ERK及p38在脂蛋白(a)促血管平滑肌细胞迁移中的作用
目的：研究ERK及p38在Lp(a)诱导的VSMC细胞骨架构建及迁移中的作用。方法：分别用不同浓度的ERK激酶抑制剂PD98059及p38激酶抑制剂SB202190抑制人VSMC内ERK及p38的活化，再用Lp(a)诱导处理后的细胞，Western blot检测P-ERK及P-p38的表达，激光共聚焦显微镜观察细胞骨架构建变化，迁移试验观察VSMC迁移数量的变化。结果：Western blot检测显示PD98059的终浓度在10μmol／L时P-ERK表达无明显变化(P＞0.05)，从20μmol／L至40μmol／L，随其终浓度的逐渐升高，P-ERK的表达逐渐降低(P＜0.01)，达到40μmol／L后，其抑制作用达到高峰；SB202190的终浓度从5μmol／L至20μmol／L，随其终浓度的逐渐升高，P-p38的表达逐渐降低(P＜0.01)，达到20μmol／L后，其抑制作用达到高峰。PD98059及SB202190分别预处理人VSMC，Lp(a)诱导20分钟后，细胞内未见明显的张力纤维、粘着斑及丝状伪足。对照组VSMC迁移细胞数为78.87±7.43个，PD98059处理组为49.20±6.47个，SB202190处理组为51.73±8.25个，PD98059处理组及SB202190处理组迁移细胞数均较正常对照组明显减少(P＜0.01)。结论：Lp(a)诱导的VSMC细胞骨架构建及细胞迁移依赖于ERK及p38的活化。
Part one Cytoskeleton reorganization and cell migration in human vascular smooth muscle cell stimulated by Lp(a)
Objectives: To study the reorganization of cytoskeleton and cell migration in human vascular smooth muscle cell (VSMC) stimulated by lipoprotein(a)[Lp(a)]. Methods: Firstly, the primary human VSMCs were cultured. Migration assays were applied to VSMC stimulated by Lp(a) of different concentrations. After VSMCs had been stimulated by Lp(a), F-actin was stained by FITC-labeled phalloidin to visualize actin-based structure including filopodia and stress fiber at different time points. And vinculin distribution of focal adhesion was visualized through indirect immunofluorescence at different time points too. These cytoskeletons of VSMC were then observed with confocal laser scanning microscope. Results: When the final concentration of Lp(a) was confined from 40μg/ml to 640μg/ml, the number of migrating VSMCs added while the final concentration was increased with significant difference (P<0.05) . The filopodia, stress fibers and focal adhesions in VSMC appeared at 10min after stimulation of Lp(a). The stress fibers and focal adhesions appeared remarkably at 20min. There was no evident change of these structures at 30min. Conclusion: Lp(a) induced the reorganization of cytoskeleton in human VSMC and stimulated cell migration of VSMC.
Part two The role of Rac1 and RhoA in cell migration of human VSMCs stimulated by Lp(a)
Objectives: To verify the effect of Rac1 and RhoA in cytoskeleton reorganization and cell migration of human VSMCs stimulated by Lp(a). Methods: The siRNA was introduced into cultured human VSMCs by lipofectamine to block the expression of
Rac1 gene. Then VSMCs were stimulated by Lp(a). Reverse-transcription polymerase chain reaction (RT-PCR) and western blot were applied to detect the efficacy of RNA interference. The cytoskeletons of these VSMCs were then observed with confocal laser scanning microscope. The number of migrated cells was determined by migration assays. C3 exoenzyme was introduced into VSMCs by lipofectamine to block the activity of RhoA. Then VSMCs were stimulated by Lp(a). The cytoskeletons of these VSMCs were then observed with confocal laser scanning microscope and the number of cells that had migrated was determined by migration assays. Results: The Rac1 siRNA demonstrated the efficiency in the blocking of Rac1, with significant difference from the control (P<0.01) . After VSMCs were stimulated by Lp(a) for 20min, there was no filopodia, stress fibers and focal adhesions in VSMC which Rac1 siRNA had been introduced into. The number of VSMCs that had migrated in the RNAi group was fewer than the control groups with significant difference (P<0.01) . After VSMCs were stimulated by Lp(a) for 20min, there was no filopodia, stress fibers and focal adhesions in VSMC which C3 exoenzyme had been introduced into. The number of VSMCs that had migrated in the C3 exoenzyme group was fewer than the control groups with significant difference (P<0.01) . Conclusion: Stimulation of VSMC cytoskeleton reorganization and migration by Lp(a) is dependent on Rac1 and RhoA.
Part three The role of ERK and p38 in cell migration of human VSMCs stimulated by Lp(a)
Objectives: To verify the effect of ERK and p38 in cytoskeleton reorganization and cell migration of human VSMCs stimulated by Lp(a). Methods: The ERK kinase inhibitor PD98059 of different concentrations and p38 kinase inhibitor SB202190 of different concentrations were applied to inhibit activation of ERK and p38 in human VSMCs. Then VSMCs were stimulated by Lp(a). Western blot were applied to detect the expression of P-ERK and P-p38. The cytoskeletons of these VSMCs were then observed with confocal laser scanning microscope. The number of cells that had migrated was determined by migration assays. Results: The expression of P-ERK was
decreasing while the final concentration of PD98059 was increased with significant difference (P<0.01) , when the final concentration of PD98059 was confined from 20μmol/L to 40μmol/L. The expression of P-p38 was decreasing while the final concentration of SB202190 was increased with significant difference (P<0.01) ,when the final concentration of SB202190 was confined from 5μmol/L to 20μmol/L. After VSMCs were stimulated by Lp(a) for 20min, there was no filopodia, stress fibers and focal adhesions in VSMCs which had been treated with PD98059 or SB202190. The number of VSMCs that had migrated in the PD98059 group and SB202190 group was fewer than the control groups with significant difference (P<0.01) . Conclusion: Stimulation of VSMC cytoskeleton reorganization and migration by Lp(a) is dependent on activation of ERK and p38.
引文
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