大蒜素对同型半胱氨酸诱导的兔血管平滑肌细胞增殖作用的实验研究
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摘要
目的:
1.观察大蒜素(Allicin)对同型半胱氨酸(Hcy)诱导的兔血管平滑肌细胞(VSMCs)增殖的影响;
2.观察Allicin对体外培养的兔VSMCs增殖细胞核抗原(PCNA)、c-myc及碱性成纤维细胞生长因子(bFGF)表达的影响;
3.为Allicin防治高同型半胱氨酸血症(HHcy)及其所致的动脉粥样硬化(AS)提供理论依据。
方法:
组织贴块法培养原代兔VSMCs,采用差速贴壁法进行纯化,取对数生长期的第4-6代细胞用于实验。由Hcy诱导建立细胞增殖模型,将细胞随机分为6组,对照组、Hcy组(只加Hcy,终浓度为1mmol/L)、Allicin组中加入Hcy(终浓度为1mmol/L)和不同浓度的Allicin(终浓度分别为10、5、2.5 ug/ml)、叶酸组加入Hcy(终浓度为1mmol/L)和叶酸(终浓度为5mmol/L)。通过MTT比色法检测细胞的增殖活度,免疫细胞化学技术检测PCNA的表达及Real-time PCR技术检测c-myc、bFGF mRNA的表达。
结果:
1、MTT检测结果显示,Hcy作用于VSMCs后,能诱导其增殖, Hcy组与对照组相比有显著性差异(P<0.01)。与Hcy组比较,不同浓度的Allicin均能明显抑制VSMCs增殖(P<0.01),并且呈剂量依赖关系,Allicin大中剂量组作用强于叶酸组(P<0.01)。
2、免疫细胞化学检测结果显示,镜下观察对照组PCNA阳性表达定位于胞核,呈现棕黄色。分析结果显示:与对照组比较,Hcy组VSMCs的PCNA的表达明显增强(P<0.01)。与Hcy组比较, Allicin大、中、小剂量组均能抑制PCNA蛋白的表达,随着Allicin浓度的增大,PCNA的表达逐渐减弱(P<0.01),且Allicin大、中剂量组的抑制作用强于叶酸组(P<0.01或P<0.05)。
3、Real-time PCR待测mRNA的表达结果显示,与对照组相比,Hcy组可明显使c-myc、bFGF表达上调,差异具有统计学意义(P<0.01),与Hcy组比较,Allicin大、中、小剂量组均能明显抑制Hcy诱导的VSMCs中的c-myc、bFGF mRNA表达,并且随Allicin浓度的增加,抑制作用增强(P<0.01), Allicin大、中剂量组的作用均强于叶酸组(P<0.05),Allicin小剂量组与叶酸组比较差异无统计学意义(P>0.05)。
结论:
Allicin能显著抑制Hcy诱导的VSMCs的增殖,表现出剂量依赖效应;其作用机制可能与其下调PCNA蛋白、下调c-myc mRNA及bFGF mRNA表达密切相关。
Objective:
1.To observe the effects of Allicin on proliferation of vascular smooth muscle cells(VSMCs) in rabbits induced by homocysteine.
2.To observe the effects of Allicin on protein expression of PCNA and mRNA expression of c-myc and bFGF.
3.To provide a theoretical basis for preventing and treating hyperhomocysteinemia and atherosclerosis with Allicin.
Methods:
VSMCs were cultured by tissue explants in vitro and pured by the difference of adherent rate.Cells in logarithmic phase from 3 to 6 generation were used to test. Proliferative models were induced by homocysteine,then the cells were divided into 6 groups:control group(no homocysteine),homocysteine group(homocysteine 1mM), different dose of Allicin groups (homocysteine 1mM + Allicin 10、5、2.5 ug/ml respectively),and folic acid group (homocysteine 1mM + 5mM folic acid ). Activity of cell proliferation was measured by MTT colorimetric method. Protein expression of PCNA was detected by immunocytochemical technique. The method of Real-time PCR was used to observe the mRNA expression of c-myc and bFGF.
Results:
1.VSMCs were incubated with homocysteine for 24 hours later,cell proliferation were significant(P<0.01).Allicin significantly decreased the proliferation of VSMCs induced by homocysteine in a dose-dependent manner(P<0.01). Folic acid also had inhibitory effect on VSMCs but was weaker than Allicn high-dose and middle-dose groups (P<0.01).2. Observed in microscope,PCNA positive expression was located in the nucleus, showing brown.The results indicated that homocysteine significantly induced the expression of PCNA in VSMCs(P<0.01).Compared with homocysteine group, Allicin groups could inhibit protein expression of PCNA . With increase of Allicin concentration, PCNA expression was decreased(P<0.01).Inhibitory effects of high and middle groups were significantly higher than folic acid group (P<0.01 or P<0.05).
3. Compared with the control group, homocysteine significantly increased the expression of c-myc and bFGF, the difference was statistically significant (P <0.01).Compared with homocysteine group,the Allicin groups had inhibitory effect on the expression of c-myc and bFGF.This effect was dependent on dose(P<0.01).Folic acid had decreased the expression ,but the effect was weaker than Allicin high-dose and middle-dose groups (P<0.05). The low dose Allicin group was get close to folic acid group with no statistical significance (P>0.05).
Conclusions:
Allicin could obviously inhibit the VSMCs proliferation induced by homocysteine. And it also presented dose-dependent.The mechanism may be related to down-regulating protein expression of PCNA, and mRNA expression of c-myc and bFGF.
1.观察大蒜素(Allicin)对同型半胱氨酸(Hcy)诱导的兔血管平滑肌细胞(VSMCs)增殖的影响;
2.观察Allicin对体外培养的兔VSMCs增殖细胞核抗原(PCNA)、c-myc及碱性成纤维细胞生长因子(bFGF)表达的影响;
3.为Allicin防治高同型半胱氨酸血症(HHcy)及其所致的动脉粥样硬化(AS)提供理论依据。
方法:
组织贴块法培养原代兔VSMCs,采用差速贴壁法进行纯化,取对数生长期的第4-6代细胞用于实验。由Hcy诱导建立细胞增殖模型,将细胞随机分为6组,对照组、Hcy组(只加Hcy,终浓度为1mmol/L)、Allicin组中加入Hcy(终浓度为1mmol/L)和不同浓度的Allicin(终浓度分别为10、5、2.5 ug/ml)、叶酸组加入Hcy(终浓度为1mmol/L)和叶酸(终浓度为5mmol/L)。通过MTT比色法检测细胞的增殖活度,免疫细胞化学技术检测PCNA的表达及Real-time PCR技术检测c-myc、bFGF mRNA的表达。
结果:
1、MTT检测结果显示,Hcy作用于VSMCs后,能诱导其增殖, Hcy组与对照组相比有显著性差异(P<0.01)。与Hcy组比较,不同浓度的Allicin均能明显抑制VSMCs增殖(P<0.01),并且呈剂量依赖关系,Allicin大中剂量组作用强于叶酸组(P<0.01)。
2、免疫细胞化学检测结果显示,镜下观察对照组PCNA阳性表达定位于胞核,呈现棕黄色。分析结果显示:与对照组比较,Hcy组VSMCs的PCNA的表达明显增强(P<0.01)。与Hcy组比较, Allicin大、中、小剂量组均能抑制PCNA蛋白的表达,随着Allicin浓度的增大,PCNA的表达逐渐减弱(P<0.01),且Allicin大、中剂量组的抑制作用强于叶酸组(P<0.01或P<0.05)。
3、Real-time PCR待测mRNA的表达结果显示,与对照组相比,Hcy组可明显使c-myc、bFGF表达上调,差异具有统计学意义(P<0.01),与Hcy组比较,Allicin大、中、小剂量组均能明显抑制Hcy诱导的VSMCs中的c-myc、bFGF mRNA表达,并且随Allicin浓度的增加,抑制作用增强(P<0.01), Allicin大、中剂量组的作用均强于叶酸组(P<0.05),Allicin小剂量组与叶酸组比较差异无统计学意义(P>0.05)。
结论:
Allicin能显著抑制Hcy诱导的VSMCs的增殖,表现出剂量依赖效应;其作用机制可能与其下调PCNA蛋白、下调c-myc mRNA及bFGF mRNA表达密切相关。
Objective:
1.To observe the effects of Allicin on proliferation of vascular smooth muscle cells(VSMCs) in rabbits induced by homocysteine.
2.To observe the effects of Allicin on protein expression of PCNA and mRNA expression of c-myc and bFGF.
3.To provide a theoretical basis for preventing and treating hyperhomocysteinemia and atherosclerosis with Allicin.
Methods:
VSMCs were cultured by tissue explants in vitro and pured by the difference of adherent rate.Cells in logarithmic phase from 3 to 6 generation were used to test. Proliferative models were induced by homocysteine,then the cells were divided into 6 groups:control group(no homocysteine),homocysteine group(homocysteine 1mM), different dose of Allicin groups (homocysteine 1mM + Allicin 10、5、2.5 ug/ml respectively),and folic acid group (homocysteine 1mM + 5mM folic acid ). Activity of cell proliferation was measured by MTT colorimetric method. Protein expression of PCNA was detected by immunocytochemical technique. The method of Real-time PCR was used to observe the mRNA expression of c-myc and bFGF.
Results:
1.VSMCs were incubated with homocysteine for 24 hours later,cell proliferation were significant(P<0.01).Allicin significantly decreased the proliferation of VSMCs induced by homocysteine in a dose-dependent manner(P<0.01). Folic acid also had inhibitory effect on VSMCs but was weaker than Allicn high-dose and middle-dose groups (P<0.01).2. Observed in microscope,PCNA positive expression was located in the nucleus, showing brown.The results indicated that homocysteine significantly induced the expression of PCNA in VSMCs(P<0.01).Compared with homocysteine group, Allicin groups could inhibit protein expression of PCNA . With increase of Allicin concentration, PCNA expression was decreased(P<0.01).Inhibitory effects of high and middle groups were significantly higher than folic acid group (P<0.01 or P<0.05).
3. Compared with the control group, homocysteine significantly increased the expression of c-myc and bFGF, the difference was statistically significant (P <0.01).Compared with homocysteine group,the Allicin groups had inhibitory effect on the expression of c-myc and bFGF.This effect was dependent on dose(P<0.01).Folic acid had decreased the expression ,but the effect was weaker than Allicin high-dose and middle-dose groups (P<0.05). The low dose Allicin group was get close to folic acid group with no statistical significance (P>0.05).
Conclusions:
Allicin could obviously inhibit the VSMCs proliferation induced by homocysteine. And it also presented dose-dependent.The mechanism may be related to down-regulating protein expression of PCNA, and mRNA expression of c-myc and bFGF.
引文
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