摘要
目的探讨 RhA/Rho 激酶在高肺血流所致肺血管结构重构过程中所起的作用。方法108只4周龄 Wistar 大鼠随机分到4个分流组、4个对照组,分流组大鼠接受左侧颈总动脉-颈外静脉分流术,对照组大鼠作假手术。在术后1、2、4、8周,分别测量右心室收缩压,作血气分析计算 Qp/Qs。肺组织切片 HE 染色光镜下观察肺血管形态学改变,测量并计算中等血管中膜厚度所占管径百分比(MT%)。免疫组化增殖细胞核抗原(PCNA)染色分析平滑肌细胞增生情况。用 TUNEL 细胞凋亡标记分析平滑肌细胞凋亡状况。pull down assay 分析 RhoA 活性,Western blot 检测 RhoA、Rho 激酶(ROCK2)的蛋白表达以及 MYPT1磷酸化程度量化分析 Rho 激酶活性。结果颈总动脉-颈外静脉分流导致肺循环血流量增加,Qp/Qs 平均值为2.26±0.35。分流组 RVSP 在术后1周和8周较对照组高(t=8.799,t=5.332,P<0.01)。与对照组相比,分流组以平滑肌细胞过度增生和肥大为特征的中等肺动脉中膜增厚,MT%升高在第4周开始出现,到第8周更为明显(t=9.192,t=11.185,P<0.01)。分流组 PCNA 阳性平滑肌细胞在第1周显著升高,第2周达最高值,与对照组相比,差异有统计学意义(t=7.213,P<0.01),到第4周下降到显著低于对照组(t=4.183,P<0.01),并持续降低,到第8周几乎观察不到增生的平滑肌细胞(t=6.152,P<0.01)。TUNEL 阳性平滑肌细胞在第2周较对照组低(t=2.418,P<0.05),并持续降低,到第8周几乎观察不到凋亡的平滑肌细胞(t=4.582,P<0.01)。RhoA 和 ROCK2 表达在第1周即高于对照组(t=6.056,t=8.411,P<0.01),第2周达高峰(t=9.342,t=10.437,P<0.01)。到第4开始下降,但是到第8周仍然显著高于对照组(t=4.743,t=4.455,P<0.01)。RhoA 和 Rho 激酶活性在第1周也显著高于对照组(t=10.246,t=19.110,P<0.01),到第4周达高峰(t=24.984,t=16.124,P<0.01),然后开始降低,但是二者到第8周仍然显著高于对照组水平(t=4.934,t=10.426,P<0.01)。结论在高肺血流所致的大鼠肺血管收缩和结构重构与 RhoA/Rho 激酶的表达和活性增强有关。
Objective High pulmonary blood flow induced pulmonary hypertension （PH） is often associated with increased vasoconstriction and deteriorating pulmonary artery remodeling, of which the exact mechanism has not been completely elucidated. The involvement of RhoA/Rho-kinase pathway has been demonstrated in the pathogenesis of hypoxia and monocrotaline induced PH. Thus the purpose of this study was to test whether RhoA/Rho-kinase pathway is involved in the process of high pulmonary flow induced pulmonary artery remodeling in rats. Methods Wistar rats aged 4 weeks in the shunt group underwent left common carotid artery-external jugular vein shunt operation, those in control group received sham-operation. At weeks 1, 2, 4 and 8 of the study, rats underwent right ventricular systolic pressure （RVSP） measurement; blood gases were analyzed to calculate Qp/Qs. The morphologic alterations of the pulmonary arteries were observed under optical microscope. The mean percentage of media wall thickness （ % MT） was also measured to assess the extent of medial wall thickness of moderate size pulmonary arteries. Proliferating smooth muscle cells （SMCs） were evaluated by proliferating cell nuclear antigen （PCNA）immunohistochemical staining. Apoptotic SMCs were detected by TUNEL method. RhoA activity in pulmonary arteries was detected using pull down assay. Rho kinase activity was quantified by the extent of MYPT1 phosphorylation with Western blotting. The expression of RhoA and Rho kinase （ROCK2） was also detected with Western blotting~ Results Carotid artery-jugular vein shunt resulted in high pulmonary blood flow, of all rats in shunted groups, the mean Qp/Qs was 2. 26 ± 0. 35, which were all considered large shunts. Compared with the control group, RVSP in shunt group increased significantly at both week 1 and week 8 （ t = 8. 799, t = 5. 332, respectively, P 〈 0. 01 ）. Compared with the control group, moderate pulmonary artery medial wall thickening characterized by SMCs hyper-proliferation and hypertrophy in shunted group was firstly appeared at week 4 and became more significant at week 8, as indicated by MT% （t = 9. 192, t = 11. 185, respectively, P 〈 0. 01 ）. Compared with the control group, the percentage of PCNA-positive SMCs in shunted group increased significantly at week 1 （ t = 2. 438, P 〈 0. 05 ）, and reached the maximal level at week 2 （t =7.213, P 〈0. 01 ）, then, it decreased to a level significantly lower than that of the control group at week 4 （t =4. 183, P 〈0. 01）, and continued to decrease to so low a level that proliferative SMCs was scarcely observed at week 8 （t = 6. 152, P 〈 0. 01 ）. The percentage of TUNEL- positive SMCs decreased significantly compared with the control group at week 2 （t = 2. 418, P 〈 0. 05 ）, and continued to decrease to a level that apoptotic SMCs was scarcely observed at week 8 （t =4. 582, P 〈 0. 01 ）. Compared with the control group, the expression of RhoA and ROCK2 increased significantly at week 1 （ t = 6. 056, t = 8. 411, respectively, P 〈 0. 01 ）, and reached the maximal level at week 2 （ t = 9. 342, t = 10. 437, respectively, P 〈 0. 01 ）, then began to decrease at week 4, however, both of them were still significantly higher than those of the control group at week 8 （ t = 4. 743, t = 4. 455, respectively, P 〈 0. 01 ）. In line with the expression of RhoA and ROCK2, both RhoA and Rho kinase activity of shunted group increased significantly compared with the control group at week 1 （t = 10. 246, t = 19. 110, respectively, P 〈 0. 01 ）, and reached the maximal level at week 4 （ t = 24. 984, t = 16. 124, respectively, P 〈 0. 01 ）, then decreased, however, both of them were still higher than those of the control group at week 8 （t = 4. 934, t = 10. 426, respectively, P 〈 0. 01 ）. Conclusion Activated RhoA/Rho-kinase pathway is associated with both high pulmonary blood flow induced acute pulmonary vasoconstriction and chronic pulmonary artery remodeling in rats.
出处
《中华儿科杂志》
CAS
CSCD
北大核心
2007年第5期387-392,共6页
Chinese Journal of Pediatrics
关键词
高血压
肺性
蛋白质丝氨酸苏氨酸激酶
胞间信号肽类和蛋白质类
肺循环
Hypertension pulmonary
Protein-serine-threonine kinases
Intercellular signaling peptides ard proteins
Pulmonary circulation
