【作者】 来丽萍；

【导师】 张继东；

【作者基本信息】 山东大学 ， 中西医结合临床， 2008， 硕士

【摘要】 目的1.观察益肾活血提取液对同型半胱氨酸诱导的兔血管平滑肌细胞增殖的影响;2.观察益肾活血提取液对NF-κzB活化、TGF-β₁、MCP-1、VCAM-1和PCNA的表达及细胞周期的影响:3.探讨NF-κB信号转导通路的调节对兔血管平滑肌细胞增殖的可能机制。方法采用组织贴块法培养原代兔血管平滑肌细胞,并通过差速贴壁法进行纯化。倒置显微镜下观察细胞形态及生长现象,SP法行a-SM-actin免疫细胞化学染色鉴定,细胞纯度达95%以上,取处于对数生长期的第3～6代细胞用于实验。建立同型半胱氨酸诱导（终浓度1mM）的细胞增殖模型,实验共分6组,空白对照组、模型组均不加益肾活血提取液,实验组细胞培养液中加入不同浓度的益肾活血提取液（终浓度分别为5000、500、50mg/L）,西药对照组加入叶酸（终浓度5mM）,继续培养24h进行实验检测。采用MTT法检测细胞的增殖活度,流式细胞术检测细胞周期的变化,免疫细胞化学染色检测NF-κBp65活化和TGF-β₁、MCP-1、VCAM-1和PCNA的蛋白表达,RT-PCR检测TGF-β₁、MCP-1、VCAM-1的mRNA表达。实验重复3次,实验数据采用统计学方法进行分析。结果1.不同浓度的益肾活血提取液作用24h后,可观察到细胞的异常增殖状态受到抑制。MTT法检测并分析其对细胞增殖的抑制作用,流式细胞仪检测细胞周期的变化,发现实验组细胞处于静止期（G₀/G₁期）的比例增加,处于DNA合成期（S期）和有丝分裂期（G₂/M期）的比例减少,这较之模型组有明显差异（P＜0.01）,且表现出剂量依赖性。2.免疫细胞化学检测显示NF-κBp65和TGF-β₁、MCP-1、VCAM-1、PCNA在平滑肌细胞内的表达及其相互关系:NF-κBp65、PCNA表达阳性产物呈棕褐色颗粒,位于细胞浆/细胞核,TGF-β₁、MCP-1、VCAM-1表达位于细胞浆,亦呈棕褐色颗粒。在益肾活血提取液作用于细胞24h后其表达水平均明显下降,且随着NF-κBp65活化的抑制,TGF-β₁、MCP-1、VCAM-1的表达也随之下调（P＜0.01）。3.RT-PCR检测分析TGF-β₁、MCP-1、VCAM-1的mRNA表达:不同浓度的益肾活血提取液作用于细胞24h后,TGF-β₁、MCP-1、VCAM-1的mRNA表达明显下调。而且,随着浓度的增加其表达呈逐渐下降的趋势,其中大、中剂量组的下调趋势明显高于西药对照组,相对光密度比值分别为0.46±0.04、0.45±0.05、0.42±0.04（P＜0.01）。结论1.益肾活血提取液可以有效地抑制NF-κB的活化,抑制平滑肌细胞的异常增殖,调节细胞周期,表现出剂量依赖效应;2.NF-κB的活性下调的同时,TGF-β₁、MCP-1、VCAM-1及PCNA等的表达也随之下调,且与NF-κB活化密切正相关;3.益肾活血提取液能有效地抑制NF-κB的活化,其对TGF-β₁、MCP-1、VCAM-1及PCNA等多种因子表达的影响可能是通过干扰基因的转录来实现的。更多还原

【Abstract】 Objectives1.To investigate the effects of Yishenhuoxue Capsule on proliferative rabbits’ vascular smooth muscle cells induced by HCY.2.To investigate the effects of Yishenhuoxue Capsule on NF-κB activation、the expression of TGF-β₁、MCP-1、VCAM-1、PCNA and cell cycles.3.To explore the potential mechanism of modulating the NF-κB signal transduction pathway on proliferative rabbits’vascular smooth muscle cells induced by HCY.MethodsVSMCs were cultured by tissue explants in vitro and pured by the difference of adherent rate.Celluar morphology and growth nature were observed with inverted microscope.After identified with a-SM-actin by SP,the purity of cells was greater than 95%,then cells in logarithmic phase from 3 generation to 6 were used to test. Proliferative cells were induced by HCY（1mM）,next they were divided into 6 groups-the control group（no HCY）、the HCY group and four treatment groups（5000、500、50mg/L YSHXC,5mM folic acid respectively）,and then tested after 24 hours culture.The proliferation of cells and cell cycles were determined by MTT and flow cytometry;the expressions of TGF-β₁、MCP-1、VCAM-1、PCNA and NF-κB activation were detected and analyzed with immunocytochemistry and RT-PCR.All tests were repeated for three times.The data was analyzed by statistics. Results1.YSHXC of different concentration could inhibit cells’proliferation.The inhibition and cell cycles detection by MTT and flow cytometry showed that,in treatment groups the number of cells at G₀/G₁ stage increased greatly,while the number at S and G₂/M stages decreased;which was different from the HCY group（P＜0.01）.And it also presented dose-dependent.2.The positive products of NF-κB activation and PCNA showed the brown particles located in cytoplasm and/or nucleus,the ones of TGF-β₁、MCP-1、VCAM-1 expression were mainly located in cytoplasm,which showed brown particles as well. They were downregulated after YSHXC treating for 24 hours.Downregulation of TGF-β₁、MCP-1、VCAM-1 expression were closely correlated with inhibition of NF-κB activation（P＜0.01）.3.RT-PCR analysis showed that,compared with the control group,VSMCs treated with YSHXC at different concentration for 24 hours indicated the decline of TGF-β₁、MCP-1、VCAM-1 expression respectively.And the decline of TGF-β₁、MCP-1、VCAM-1 expression of the YSHXC higherdose and middledose groups was superior to that of the folic acid group 0.46±0.04、0.45±0.05、0.42±0.04（P＜0.01）.Conclusions1.YSHXC can effectively and dose-dependently inhibit the NF-κB activation, reduce the abnormal proliferation of VSMCs,and adjust the cell cycles.2.TGF-β₁、MCP-1、VCAM-1、PCNA expression will downregulate accompanied with the inhibition of NF-κB activation,which were closely correlated.3.Inhibition of NF-κB activation by YSHXC may affect the expressions of TGF-β₁、MCP-1、VCAM-1、PCNA through interfering the gene transcription action.更多还原