【作者】 王岩；

【导师】 周亚滨；

【作者基本信息】 黑龙江中医药大学 ， 中医内科学， 2010， 博士

【摘要】 目的:通过观察养心汤含药血清对过氧化氢诱导人脐静脉内皮细胞氧化损伤模型差异基因及ST3GAL2mRNA表达的影响,探讨其内皮保护机制,为早期防治冠心病不稳定型心绞痛提供客观依据。方法:采用过氧化氢体外诱导人脐静脉内皮细胞氧化损伤的方法,建立人脐静脉内皮细胞氧化损伤模型。实验一分为①空白血清组;②模型组;③养心汤含药血清2%组;④养心汤含药血清4%组;⑤养心汤含药血清6%组;⑥养心汤含药血清8%组。倒置显微镜下观察细胞形态、密度,MTT法测定各组细胞增殖活性。实验二分为①空白组;②模型组;③养心汤含药血清组,应用Affymetrix人基因组U133+2.0芯片观察养心汤含药血清对过氧化氢诱导的人脐静脉内皮细胞氧化损伤模型的差异基因表达的影响,并采用实时定量PCR方法对人脐静脉内皮细胞氧化损伤模型ST3GAL2mRNA的表达进行验证。结果:1.各组人脐静脉内皮细胞形态学改变:在倒置显微镜下可观察到,正常内皮细胞排列紧密,呈铺路石状。过氧化氢作用2h后,细胞变圆,开始脱落,细胞间隙增宽,密度明显降低,细胞呈梭形,细胞核折光性增强。养心汤含药血清2%、4%、6%、8%组细胞形态有所改善,轮廓相对融合。其中养心汤含药血清6%、8%组细胞变形、皱缩的程度减轻最为明显;2.与空白血清组比较,模型组细胞光密度值、增殖率均降低,其差异显著,具有统计学意义(P均<0.01)。与模型组比较,养心汤含药血清2%组细胞光密度值、增殖率均升高,差异无统计学意义(P均>0.05),养心汤含药血清4%、6%、8%组细胞光密度值、增殖率均升高,差异显著,具有统计学意义(P均<0.01)。与养心汤含药血清6%组比较,养心汤含药血清2%、4%组的细胞光密度值、增殖率均降低,差异显著,具有统计学意义(P均<0.01);养心汤含药血清8%组的细胞光密度值、增殖率均降低,差异无统计学意义(P均>0.05);3.模型组与空白组比较表达上调,养心汤含药血清组与模型组比较表达下调的基因有23个,模型组与空白组比较表达下调,养心汤含药血清组与模型组比较表达上调的基因有12个,经MAS分析后,养心汤含药血清干预人脐静脉内皮细胞氧化损伤模型差异基因的功能主要集中在与核酸结合、与蛋白激酶结合、蛋白质合成和转运、血小板活化、细胞黏附等方面;4.养心汤含药血清组ST3GAL2mRNA表达量为模型组的0.375倍,与基因芯片检测结果基本一致。结论:1.养心汤含药血清可以减轻过氧化氢对人脐静脉内皮细胞增殖的抑制作用;2.养心汤含药血清干预人脐静脉内皮细胞氧化损伤模型差异基因的功能主要集中在与核酸结合、与蛋白激酶结合、蛋白质合成和转运、血小板活化、细胞黏附等方面;3.养心汤含药血清可明显下调人脐静脉内皮细胞氧化损伤模型ST3GAL2mRNA表达水平,这可能是其保护内皮细胞损伤,防治冠心病不稳定型心绞痛的机制之一。更多还原

【Abstract】 Objective:To observe the effects the formula of the serum with Yangxin Decoction on the oxidative damage of human umbilical vein endothelial cell’different genes expressions.To investigate its mechanics, then to provide academic warranties for preventing and curing the atherosc- lerosis early.Methods:Establishing the Cell models of the oxidative damage of human umbilical vein endothelial cell with the method of interferencing Hydrogen peroxide to human umbilical vein endothelial cell. Deviding the human umbilical vein endothelial cell into six groups random.①The blank control group ;②The model group;③the serum with Yangxin Decoction 2% group;④the serum with Yangxin Decoction 4% group;⑤the serum with Yangxin Decoction 6% group;⑥the serum with Yangxin Decoction 8% group.Observe cellular form, density Under inverted microscope. examine cellular viability with MTT.To observe the effects the formula of the serum with Yangxin Decoction on the oxidative damage of human umbilical vein endothelial cell’different genes expressions. To examine the expression of ST3GAL2mRNA with RT-PCR method.Result:1.The morphologic change of human umbilical vein endothel- ial cell: Normal umbilical vein endothelial cell’s closely arranged, cobble- stone-like under the inverted microscope. The cell tunes to be round, falls off and the intercellular space broadens, the density reduces obviously, the cell looks like spindle-shaped, nucleus diffraction enhances after 100μmol L-1 hydrogen peroxide affects 2h;2.Model group was significantly lower than the blank control group in OD, statistically significant difference (P<0.01).Compared with model group, the serum with Yangxin Decoction 2% group has no significant difference(P>0.05) , the serum with Yangxin Decoction 4%、6%、8% group have significant differences(P<0.01). Compared with the serum with Yangxin Decoction 6%, the serum with Yangxin Decoction 2%、4%group have significant differences(P<0.01),the serum with Yangxin Decoction 8% group has no significant difference(P> 0.05);3.Model group compared with the blank control group upregulation, the serum with Yangxin Decoction compared with the model group downreg- ulation12 genes,Model group compared with the blank control group down- regulation, the serum with Yangxin Decoction compared with the model gro- up upregulation23 genes;4. The serum with Yangxin Decoction’expression of ST3GAL2mRNA 0.375 times for the model group,this is consistent with microarray results.Conclusions:1.The serum with Yang xin Decoction can reduce the inhibiti- on in which hydrogen peroxide effect human umbilical vein endothelial cell proliferation;2.The serum with Yangxin Decoction’different gene functions mainly in DNA binding,protein synthesis and transport,kinase,Platelet acti- vation,cell adhesion,etc;3.The serum with Yangxin Decoction can significa- ntly lower the oxidative injury model’ST3GAL2mRNA expression,which may be its function mechanism of its preventing and curing atherosclerosis.更多还原