【作者】 呼海娟；

【导师】 韩梅；

【作者基本信息】 河北医科大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 目的:血管平滑肌细胞(vascular smooth muscle cell, VSMC)增殖是血管再狭窄的主要病理基础。黄芩苷(baicalin)是从中草药黄芩中分离出来的一种黄酮类化合物。以往研究发现,黄芩苷对肿瘤细胞的增殖具有明显的抑制作用。但其是否影响血管内皮损伤所诱导的血管内膜增生尚不清楚。本研究从以下几方面探讨黄芩苷对血管新生内膜形成的影响及其作用机制。方法:1 VSMC的培养:按贴块法分离培养VSMC,取3～6代细胞进行实验。传代培养的VSMC生长到80%～90%汇合时,给予不同浓度黄芩苷(5、10、20、40μmol/L)预处理。2 Western blot分析:取等量蛋白的提取液上样,进行SDS-PAGE。电转移至PVDF膜上。随后与相应的一抗及二抗反应,用化学发光法检测抗原抗体结合区带。用数码成像分析系统软件对结果进行定量分析。3球囊损伤模型的复制及分组将SD大鼠(250~300g)常规麻醉消毒,沿颈部正中线切口,暴露左侧颈外动脉,在颈外动脉剪一楔形切口,将球囊导管自切口插入至胸腹主动脉远心端,充盈球囊后反复回拉三次,退出,结扎颈外动脉,关闭切口。实验动物分假手术组、球囊损伤组、黄芩苷组。4标本的处理于术后14天从股动脉放血处死大鼠,分离颈总动脉,制备病理切片。结果:1黄芩苷抑制PDGF诱导的VSMC增殖MTT分析结果表明,黄芩苷能以浓度(5、10、20、40μmol/L)依赖的方式抑制VSMC增殖,具有明显的量-效关系。2黄芩苷对VSMC增殖标志基因PCNA表达的影响免疫细胞化学和Western blot分析显示,正常细胞中PCNA呈低水平表达,PDGF (10 ng/ml)刺激后PCNA水平大幅度升高,而药物预孵育则使PCNA水平下降。提示黄芩苷具有抑制PDGF诱导的VSMC增殖的作用。3黄芩苷抑制PDGF激活的PDGFR/MEK/ERK1/2信号通路Western blot结果显示,PDGF刺激细胞15 min,PDGFR磷酸化水平明显升高;而经黄芩苷预处理的VSMC,磷酸化的PDGFR呈浓度依赖性的降低;进而对其下游的MEK、ERK1/2活性进行分析,发现MEK和ERK1/2磷酸化程度也随着药物浓度的增加逐渐降低;但磷酸化的JNK、p38、Akt不受药物处理的影响。提示黄芩苷是通过特异性抑制MEK/ERK1/2通路活化来抑制PDGF诱导的VSMC的增殖。4黄芩苷抑制球囊损伤后血管内膜的增生球囊损伤术后14天,黄芩苷组血管内膜的增生程度较球囊损伤组明显减轻,两组的血管内膜/中膜(I/M)厚度比分别为0.43±0.04和2.36±0.15,具有显著性差异(P < 0.01)。表明黄芩苷可以明显抑制球囊损伤诱导的血管内膜增生。5黄芩苷抑制新生内膜中PCNA表达形态学分析显示:正常血管内膜中仅有少量PCNA表达,球囊损伤14天后,血管中PCNA阳性的细胞数明显增多;而黄芩苷可明显抑制PCNA的表达,与模型组相比PCNA阳性的细胞数减少了53.4 %,具有显著性差异(P < 0.01)。提示黄芩苷可抑制血管细胞的增殖活性。6黄芩苷抑制球囊损伤诱导的ICAM-1和VCAM-1的表达免疫组织化学染色显示:正常血管内膜中可见极少量的ICAM-1和VCAM-1着色颗粒。球囊损伤14天后,两种蛋白阳性染色的细胞数及单细胞染色强度均较正常组显著增加。而黄芩苷能够显著抑制球囊损伤诱导的ICAM-1和VCAM-1的表达,与模型组相比阳性染色的细胞数分别降低了55.8±6.9%和57.2±5.5%,具有显著性差异(P<0.01,P<0.01)。结论:1黄芩苷抑制PDGF诱导的VSMC增殖。2黄芩苷抑制VSMC增殖的机制与阻断PDGFR/MEK/ERK1/2信号通路有关。3黄芩苷可预防内皮损伤所致的血管内膜增生和管腔狭窄。4黄芩苷抑制血管内膜增生与其抑制血管细胞增殖和粘附分子表达有关。更多还原

【Abstract】 It is known that the proliferation of vascular smooth muscle cells (VSMC) plays an important role in the pathogenesis of restenosis. Baicalin is a major flavonoid extracted from the traditional Chinese medicinal herb Scutellaria baicalensis Georgi. Baicalin has been used in the treatment of breast, hepatocellular, pancreatic, and prostatic cancers. However, its effects on the neointimal hyperplasia remain unclear. Therefore, the present study was designed to elucidate the effect of baicalin on the neointimal hyperplasia after balloon injury and the related mechanism.Methods1 VSMC culture5-week-old male SD rats were selected. The VSMCs from rat aorta were isolated and were cultured in DMEM medium containing 10%FBS. The cells used for the experiment were passage 3 ~ 6. The cells grown into 80%~90% confluences were serum-starved for 24 hours. Then the VSMC was treated with baicalin using different doses (5, 10, 20, 40μmol/l) for 24 h.2 Western blot analysisEqual amounts of protein extracts from VSMC were separated on 10% SDS-PAGE, and then blotted onto PVDF membrane. The membrane was immunologically stained with specific antibodies. The results were analyzed by digital imaging system.3 Establishment of the animal modelSD rats were randomly divided into three groups, including sham group, injured group, baicalin group. Rats were anesthetized with urethane. The aorta and carotid artery were de-endothelialized with balloon catheter. Briefly, the catheter was pushed from left carotid artery into the aorta down to the level of the renal arteries three times with a 2F Fogarty catheter, and then recovered the blood stream.4 Preparation of experimental specimenThe rats were killed at 14 days after de-endothelium. The carotid arteries were separated for preparation of sections. Sections were stained with hematoxylin and eosin to detect the neointimal hyperplasia. The microscopical pictures were analyzed using a computer assisted image analyzer, and the thickness of neointima was measured. Furthermore, the effects of baicalin on the expression of PCNA, ICAM-1 and VCAM-1 were analyzed by immunohistochemistry.Results1 The role of baicalin in VSMC proliferation induced by PDGFThe results of MTT assays showed that baicalin (5, 10, 20, 40μmol/L) treatment resulted in a significant reduction of VSMC proliferation in a dose-dependent manner.2 The effect of baicalin on PCNA expression induced by PDGFThe results of immunocytochemistry and Western blot analysis showed that expression of PCNA was rarely detected in control group. PDGF (10 ng/ml) stimulation resulted in significant increase in PCNA expression. However, the amount of PCNA was significantly reduced in the baicalin-treated group, suggesting that baicalin inhibits the proliferation of VSMC induced by PDGF.3 Baicalin markedly inhibits the PDGFR/MEK/ERK1/2 signaling pathway activated by PDGFWestern blot analysis showed that PDGF stimulation resulted in significant increase in the phosphorylation of PDGFR, MEK and ERK1/2, compared with control group. However, the phosphorylation of PDGFR, MEK and ERK1/2 induced by PDGF was significantly reduced in baicalin-treated group.4 Baicalin inhibits neointimal hyperplasia induced by balloon injury in carotid arteries of ratAt 14 days after balloon injury, the injured group showed neointimal formation in carotid arteries of rat. However, baicalin significantly reduced neointimal hyperplasia and I/M ratio (I/M ratio, baicalin versus injured, 0.43±0.04 versus 2.36±0.15, p < 0.05) compared with the injured group.5 Baicalin inhibits the expression of PCNA induced by balloon injury in carotid arteries of ratImmunohistochemical staining showed that the expression of PCNA was rarely detected in in carotid arteries of sham group. However, the number of PCNA positive cells in injured group was significantly increased. However, the expression of PCNA in carotid arteries injured by balloon was significantly reduced by 53.4 % (P<0.01) at 14 day in baicalin-treated group.6 Baicalin inhibits the expression of ICAM-1 and VCAM-1 induced by balloon injury in carotid arteries of ratImmunohistochemistry results showed that the expression of ICAM-1 and VCAM-1 in carotid arteries was not detected in sham group. Balloon injury resulted in significant increase in ICAM-1 and VCAM-1 expression, compared with sham-operated rats. Staining results also showed that the expression of ICAM-1 and VCAM-1 induced by balloon injury was significantly reduced by 55.8% and 57.2% (P<0.01) in baicalin-treated group.Conclusions1 Baicalin inhibits the proliferation of VSMC induced by PDGF.2 Baicalin blocks PDGFR-MEK-ERK1/2 signaling pathway activated by PDGF in VSMCs.3 Baicalin inhibits neointimal hyperplasia via inhibiting vascular cell proliferation and adhesion molecules expression induced by balloon injury.更多还原