冠心康对同型半胱氨酸致血管内皮细胞损伤的保护机制的实验研究
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摘要
目的(1)通过对人脐静脉内皮细胞(HUVEC)的原代培养、传代及冻存与复苏,以及含中药冠心康药物血清的制备,为进一步研究中药防治AS的机制提供充足的实验材料。(2)通过观察同型半胱氨酸对HUVEC的刺激作用,探讨Hcy诱导HUVEC细胞凋亡的作用机制。(3)观察冠心康药物血清干预Hcy作用的HUVEC后各项指标的变化,探讨中药冠心康防治Hcy致AS的作用机制。
方法(1)应用胰酶消化法,将人脐静脉内皮细胞分离下来,并用含20%胎牛血清的DMEM培养液进行培养,在细胞达到80%融合后,消化传代,并将获得的细胞进行鉴定与冻存;应用中药血清药理学的方法,制备含不同中药药物浓度的药物血清。(2)将获得的HUVEC随机分为6组,即为空白对照组、0.01mmol/L Hcy组、0.1mmol/L Hcy组、1.0mmol/L Hcy组、2.0mmol/L Hcy组、3.0mmol/L Hcy组,然后分别在3h、6h、12h、24h测定细胞的凋亡率、线粒体膜电位及作用24h时内质网伴侣蛋白Grp78蛋白表达水平的变化。(3)将获得的HUVEC随机分为5组,分别为:空白对照组、模型对照组、冠心康低浓度组、冠心康中浓度组、冠心康高浓度组;在冠心康作用2h后,每组细胞分别加入3.0 mmol/L的Hcy,分别作用3h、6h、12h和24h,观察血管内皮细胞的凋亡率、线粒体膜电位、Grp78蛋白表达水平的不同以及细胞内ROS和LOX-1蛋白表达的变化。
结果(1)经过鉴定,证明获得的细胞为HUVEC,细胞生长状态良好,冻存复苏后的细胞其生长情况与未冻存的细胞无差别;并制备出含不同药物浓度的中药血清(。2)在0.1mmol/L Hcy作用6h后,细胞凋亡率明显升高,线粒体膜电位降低明显,其结果随Hcy浓度的增加、作用时间的延长而逐渐差异显著,与空白对照组比较有显著差异(P<0.05);Grp78的蛋白表达在0.1mmol/L开始表达上调,并呈浓度依赖性,于3.0mmol/L,达到高峰。(3)模型组细胞细胞凋亡率显著增高,线粒体膜电位下降显著,Grp78的蛋白表达明显上调,内皮细胞内ROS升高,LOX-1表达上调,与空白组比较有统计学意义(P<0.05);在中、高浓度的冠心康干预下,血管内皮细胞的细胞凋亡率显著下降,线粒体膜电位下降减低,Grp78的蛋白表达降低,ROS生成减少,LOX-1的蛋白表达下调,与模型组比较有显著差异(P<0.01),且随着作用时间的延长,作用越显著。
结论(1)应用酶消化法,可以获得HUVEC,并可以通过技术将其进行冻存与复苏;应用血清药理学的原理,能制备含不同浓度的中药药物血清,为进一步研究打下基础。(2)Hcy能够诱导血管内皮细胞凋亡及Grp78蛋白水平的表达上调,其结果呈浓度、作用时间依赖性,说明其作用机制可能是引起内质网应激,促进内质网内未折叠蛋白堆积,内质网伴侣蛋白表达上调所致。(3)冠心康能够拮抗同型半胱氨酸对血管内皮细胞的损伤作用,且与冠心康的浓度、作用时间呈正性依赖关系;其作用机制可能是冠心康通过抑制同型半胱氨酸诱导的内质网应激,从而降低内皮细胞的凋亡率,减少细胞内ROS的产生,减轻同型半胱氨酸所致的脂质代谢紊乱而实现的。
Methods:(1) In the strict aseptic conditions, isolate human umbilical vein endothelial cells with Trypsin from newborn umbilical cord,then culture the cell with DMEM medium containing 20% fetal bovine serum.When the cells reach 80% confluence, digeste passaged、identification and cryopreservation;Application of Chinese medicine serum pharmacology, Prepare drug serum containing different concentrations of medicine drugs. (2) Divide the HUVEC into 6 groups randomly, each group of cells separately using 0.0mmol/L (i.e. for blank control group), 0.01 mmol / L, 0.5mmol / L, 1.0mmol / L, 2.0mmol / L, 3.0mmol / L Hcy role in HUVEC, and then at 3 h, 6h, 12h, 24h rate of cell apoptosis percentage(PI method), mitochondrial membrane potential (JC-1) and protein expression of the endoplasmic reticulum chaperone protein--Grp78 changes at 24h.(3) Divide the HUVEC into 5 groups randomly: control group、model control group、Guanxinkang low concentrations group、Guanxinkang middle concentration group、high concentration groups. Actting for 2h, added 3.0 mmol / L Hcy in every group of cells.At 6 h and 12h and 24h observe vascular endothelial cell apoptosis percentage , mitochondrial membrane potential, Grp78 protein expression as well as to different levels of ROS and LOX-1 changes.
Results:(1) With the identification, the cells have proofed it is HUVEC. The condition of the cell growth is good, and save the recovery after frozen the cell ,there has no difference from the cell which has recovered with the cell which has not saved frozen frozen. And we Have prepared drug serum with different concentrations of Chinese medicine.(2) after 0.1mmol/L Hcy affects 6h, apoptosis was significantly increased , mitochondrial membrane potential decreased significantly. The result of increasing concentrations of Hcy, there are gaps in the control group significant difference (P <0.05)as well as the extension of time.when Hcy reached 5.0mmol / L at 24h it reach a peak; Grp78 protein expression in 0.5 mmol / L increase the expression, and a concentration -dependent manner, at 3.0 mmol / L reached a peak.(3) With model group compared with control group, has significantly increased , mitochondrial membrane potential has decreased significantly and Grp78 protein expression has significantly increased (P <0.05). ROS and LOX-1 in endothelial cells has both increased.GuanxinKang high concentration of coronary intervention, the apoptosis percentage significantly decreased, mitochondrial membrane potential declined the reduce and the protein expression of Grp78 reduced.ROS and LOX-1 production decreased.Compared with the model group, there were significantly different ( P <0.01). And with the extension of time, the more significant roled.
Conclusions: (1) We can obtained HUVEC with the Tryption digestion,and can be carried out through technical cryopreservation and recovery.Application of the principle of serum pharmacology,we can prepare drug serum of traditional Chinese medicine containing different concentrat -ions.These builds the foundation for further study. (2) Hcy can induce apoptosis in vascular endothelial cells and upward the level of protein expression of Grp78.And its result assumes the density or the response time dependence.The mechanism of that may be that Hcy can cause endoplasmic reticulum stress of VEC, and promote endoplasmic reticulum, so the unfolding protein accumulated, and the endoplasmic reticulum protein expression increased by the partner. (3) Guanxinkang can antag -onistic the injury induced by Hcy of the vascular endothelial cell. And its result assumes the density or the response time dependence of the positive relationship.The mechanism may be that Guanxinkang can inhibite the endoplasmic reticulum stress induced by homocysteine, thereby decrease endothelial cell apoptosis percentage, reduce the oxidative stress induced homocysteine ,and be achievable finally.
方法(1)应用胰酶消化法,将人脐静脉内皮细胞分离下来,并用含20%胎牛血清的DMEM培养液进行培养,在细胞达到80%融合后,消化传代,并将获得的细胞进行鉴定与冻存;应用中药血清药理学的方法,制备含不同中药药物浓度的药物血清。(2)将获得的HUVEC随机分为6组,即为空白对照组、0.01mmol/L Hcy组、0.1mmol/L Hcy组、1.0mmol/L Hcy组、2.0mmol/L Hcy组、3.0mmol/L Hcy组,然后分别在3h、6h、12h、24h测定细胞的凋亡率、线粒体膜电位及作用24h时内质网伴侣蛋白Grp78蛋白表达水平的变化。(3)将获得的HUVEC随机分为5组,分别为:空白对照组、模型对照组、冠心康低浓度组、冠心康中浓度组、冠心康高浓度组;在冠心康作用2h后,每组细胞分别加入3.0 mmol/L的Hcy,分别作用3h、6h、12h和24h,观察血管内皮细胞的凋亡率、线粒体膜电位、Grp78蛋白表达水平的不同以及细胞内ROS和LOX-1蛋白表达的变化。
结果(1)经过鉴定,证明获得的细胞为HUVEC,细胞生长状态良好,冻存复苏后的细胞其生长情况与未冻存的细胞无差别;并制备出含不同药物浓度的中药血清(。2)在0.1mmol/L Hcy作用6h后,细胞凋亡率明显升高,线粒体膜电位降低明显,其结果随Hcy浓度的增加、作用时间的延长而逐渐差异显著,与空白对照组比较有显著差异(P<0.05);Grp78的蛋白表达在0.1mmol/L开始表达上调,并呈浓度依赖性,于3.0mmol/L,达到高峰。(3)模型组细胞细胞凋亡率显著增高,线粒体膜电位下降显著,Grp78的蛋白表达明显上调,内皮细胞内ROS升高,LOX-1表达上调,与空白组比较有统计学意义(P<0.05);在中、高浓度的冠心康干预下,血管内皮细胞的细胞凋亡率显著下降,线粒体膜电位下降减低,Grp78的蛋白表达降低,ROS生成减少,LOX-1的蛋白表达下调,与模型组比较有显著差异(P<0.01),且随着作用时间的延长,作用越显著。
结论(1)应用酶消化法,可以获得HUVEC,并可以通过技术将其进行冻存与复苏;应用血清药理学的原理,能制备含不同浓度的中药药物血清,为进一步研究打下基础。(2)Hcy能够诱导血管内皮细胞凋亡及Grp78蛋白水平的表达上调,其结果呈浓度、作用时间依赖性,说明其作用机制可能是引起内质网应激,促进内质网内未折叠蛋白堆积,内质网伴侣蛋白表达上调所致。(3)冠心康能够拮抗同型半胱氨酸对血管内皮细胞的损伤作用,且与冠心康的浓度、作用时间呈正性依赖关系;其作用机制可能是冠心康通过抑制同型半胱氨酸诱导的内质网应激,从而降低内皮细胞的凋亡率,减少细胞内ROS的产生,减轻同型半胱氨酸所致的脂质代谢紊乱而实现的。
Methods:(1) In the strict aseptic conditions, isolate human umbilical vein endothelial cells with Trypsin from newborn umbilical cord,then culture the cell with DMEM medium containing 20% fetal bovine serum.When the cells reach 80% confluence, digeste passaged、identification and cryopreservation;Application of Chinese medicine serum pharmacology, Prepare drug serum containing different concentrations of medicine drugs. (2) Divide the HUVEC into 6 groups randomly, each group of cells separately using 0.0mmol/L (i.e. for blank control group), 0.01 mmol / L, 0.5mmol / L, 1.0mmol / L, 2.0mmol / L, 3.0mmol / L Hcy role in HUVEC, and then at 3 h, 6h, 12h, 24h rate of cell apoptosis percentage(PI method), mitochondrial membrane potential (JC-1) and protein expression of the endoplasmic reticulum chaperone protein--Grp78 changes at 24h.(3) Divide the HUVEC into 5 groups randomly: control group、model control group、Guanxinkang low concentrations group、Guanxinkang middle concentration group、high concentration groups. Actting for 2h, added 3.0 mmol / L Hcy in every group of cells.At 6 h and 12h and 24h observe vascular endothelial cell apoptosis percentage , mitochondrial membrane potential, Grp78 protein expression as well as to different levels of ROS and LOX-1 changes.
Results:(1) With the identification, the cells have proofed it is HUVEC. The condition of the cell growth is good, and save the recovery after frozen the cell ,there has no difference from the cell which has recovered with the cell which has not saved frozen frozen. And we Have prepared drug serum with different concentrations of Chinese medicine.(2) after 0.1mmol/L Hcy affects 6h, apoptosis was significantly increased , mitochondrial membrane potential decreased significantly. The result of increasing concentrations of Hcy, there are gaps in the control group significant difference (P <0.05)as well as the extension of time.when Hcy reached 5.0mmol / L at 24h it reach a peak; Grp78 protein expression in 0.5 mmol / L increase the expression, and a concentration -dependent manner, at 3.0 mmol / L reached a peak.(3) With model group compared with control group, has significantly increased , mitochondrial membrane potential has decreased significantly and Grp78 protein expression has significantly increased (P <0.05). ROS and LOX-1 in endothelial cells has both increased.GuanxinKang high concentration of coronary intervention, the apoptosis percentage significantly decreased, mitochondrial membrane potential declined the reduce and the protein expression of Grp78 reduced.ROS and LOX-1 production decreased.Compared with the model group, there were significantly different ( P <0.01). And with the extension of time, the more significant roled.
Conclusions: (1) We can obtained HUVEC with the Tryption digestion,and can be carried out through technical cryopreservation and recovery.Application of the principle of serum pharmacology,we can prepare drug serum of traditional Chinese medicine containing different concentrat -ions.These builds the foundation for further study. (2) Hcy can induce apoptosis in vascular endothelial cells and upward the level of protein expression of Grp78.And its result assumes the density or the response time dependence.The mechanism of that may be that Hcy can cause endoplasmic reticulum stress of VEC, and promote endoplasmic reticulum, so the unfolding protein accumulated, and the endoplasmic reticulum protein expression increased by the partner. (3) Guanxinkang can antag -onistic the injury induced by Hcy of the vascular endothelial cell. And its result assumes the density or the response time dependence of the positive relationship.The mechanism may be that Guanxinkang can inhibite the endoplasmic reticulum stress induced by homocysteine, thereby decrease endothelial cell apoptosis percentage, reduce the oxidative stress induced homocysteine ,and be achievable finally.
引文
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