兰州大学机构库 >学院待认领
当归红芪超滤膜提取物对过氧化氢诱导心肌细胞氧化损伤的保护作用
Alternative TitleProtective Effects of the Ultra-Filtration Extract Mixture from Angelica Sinensis and Hedysarum Polybotrys on Hydrogen Peroxide-Induced Oxidative Damage in Cardiomyocytes
马燕花
Thesis Advisor赵健雄 ; 李应东
2010-05-19
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name博士
Keyword当归红芪超滤膜提取物 氧化应激 心肌细胞 细胞凋亡
Abstract一、目的 1、运用超滤膜技术对中药合剂进行分离、纯化。 2、研究当归红芪超滤膜提取物抗心肌细胞氧化损伤的作用。 3、研究当归红芪超滤膜提取物抗心肌细胞凋亡的作用。 4、研究当归红芪超滤膜提取物对氧应激状态下心肌细胞热休克蛋白70表达的影响。 5、为当归红芪超滤膜提取物的临床应用提供实验依据。 二、方法 1、应用陶瓷膜微滤和聚丙烯腈(PAN)中空纤维超滤膜精制当归、红芪合剂(1:5)水提物。 2、Wistar乳鼠心肌细胞原代培养,造模倒置相差显微镜观察心肌细胞形态学改变;MTT法检测心肌细胞细胞活力改变;Annexin V-FITC/PI双染法标记凋亡心肌细胞,采用流式细胞术检测各组心肌细胞凋亡指数。 4、应用半定量逆转录聚合酶链反应(RT-PCR)技术和蛋白印(Western- blotting)技术测定凋亡相关基因蛋白Hsp70、Caspase-3、Bcl-2、Bax的表达。 5、用生化学技术检测氧化应激状态下心肌细胞相关酶类如SOD、LDH、CK、MDA、MPO的变化。 三、结果 1、经紫外分光光度计测定,10万分子量当归红芪超滤膜提取物多糖含量接近单剂含量。 2、倒置相差光学显微镜下观察:正常心肌细胞伸出伪足互相融合成片,细胞核折光性好,搏动具有整体性、协调性和规律性(98.2±13.4次/min)。H2O2损伤组细胞皱缩,细胞核暗淡,搏动明显减弱(20.1±3.3次/min),部分停搏,搏动幅度强弱不一,搏动频率与正常对照组相比较差异具有显著性(P<0.01)。当归红芪超滤物高、中、低剂量干预组搏动频率略减低,但高于氧化损伤组(P<0.01)。 3、MTT法检测心肌细胞的存活率:氧化损伤模型组心肌细胞与正常对照组(89.7%)比较具有统计学意义(P<0.01)。当归红芪超滤物干预组心肌细胞的受损显著改善,存活率显著提高,高、中、低剂量组分别为77.6%、64.4%、43.9%,与氧化损伤组比较差异显著(P<0.01)。 4、生化学检测:H2O2处理使细胞膜的通透性增加,培养介质中LDH、CK的含量增加,与正常对照组比较,具有显著性差异(P<0.01)。当归红芪超滤物可显著抑制H2O2所致心肌细胞膜通透性的改变,高剂量组、中剂量组、低剂量组均能减少LDH、CK的外漏,与模型组比较差异显著(P<0.01)。H2O2损伤组细胞内MDA、MPO含量增加、SOD活力降低,与正常对照组比较,差异显著(P<0.01)。当归红芪超滤物可显著降低心肌细胞内MDA、MPO的含量,提高SOD活力,高剂量组、中剂量组、低剂量组与模型组比较均具有显著性差异(P<0.01);且呈剂量依赖关系,随超滤物浓度的增加LDH、CK、MDA、MPO含量逐渐减少,SOD活力逐渐增加。 5、RT-PCR检测结果:正常对照组Hsp70、Caspase-3、Bcl-2、Bax mRNA呈低水平表达,模型组Hsp70、Caspase-3、Bax mRNA表达量显著增加,Bcl-2 mRNA表达量显著降低,与正常对照组相比差异有统计学意义(P<0.05);与模型组比较,各超滤物干预组Hsp70 mRNA呈过度表达,Bcl-2 mRNA表达量显著增加,而Bax、Caspase-3 mRNA表达量显著降低,差异有统计学意义(P<0.05)。 6、Western-blotting检测结果:400μmol•L-1 H2O2处理心肌细胞6h后,模型组心肌细胞中Hsp70、Bax蛋白的表达明显增加,Bcl-2蛋白的表达明...
Other AbstractObjective 1.To separate and purify traditional Chinese medicine mixture through the use of ultrafiltration membrane technology. 2. To study anti-oxidative damage mechanism of UFE-AH. 3. To study anti-apoptotic mechanism of UFE-AH. 4. To study the effect of UFE-AH on expression of Hsp70 in Cardiomyocyte. 5. To provide experimental theoretic foundation for clinical applications of UFE-AH. Methods 1. Refined the mixture of angelica sinensis and hedysarum polybotrys(1:5)using ceramic membrane and PAN of the hollow fiber . 2. Primary culture of neonatal ratcardiomyocytes. 3. Constructing oxidative stress mode by using peroxide. 4. The expression of apoptosis-related gene (Hsp70, Caspase-3, Bcl-2 and Bax) were detected by RT-PCR and Western-blotting. 5. The changes of enzymes in oxidative stress myocardial cells were detected by biochemical technology,such as SOD, LDH, CK, MDA and MPO. RESULETS 1. The UFE-AH (100,000 molecular weight) was found that it did not contain ferulic acid, and polysaccharide concentrations of its mixture closed to single-dose levels by UV spectrophotometer and high performance liquid chromatography. 2.Observing myocardial cell morphology under inverted phase contrast light microscopy: Normal myocardial cells extended pseudopods with each other and blended them into films. The refractive indexes of nuclears were good. Cells beat with integrity, coherence and regularity(98.2±13.4 times /min). In H2O2 injury group, cells shrinked, nuclears were bleak, and the beats of cells were significantly decreased(20.1±3.3 times /min) and some stopped beating. The pulse amplitude of cells changed of varying intensity and their beating rates had significantly difference, compared with the normal control group(P<0.01). In UFE-AH intervention group of high, medium and low-dose, myocardial pulsating frequencies were 86.9±8.4, 71.1±6.7 and 43.7±9.6 times/min, and cell pseudopods were thinner than those in control group, and beat frequency was slightly reduced, but higher than those in group of oxidative damage. 3. Detecting the survival rate of myocardial cells by MTT assay: In oxidative injury model group, myocardial cells were severely damaged, cell survival rates were decreased significantly(29.5%) and there were statistically significant compared with normal control group (89.7%, P<0.01). In UFE-AH intervention group the damages of myocardial cells were significantly improved. In high, middle and low-dose group the cell surviv...
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Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/223650
Collection学院待认领
Affiliation临床医学院
Recommended Citation
GB/T 7714
马燕花. 当归红芪超滤膜提取物对过氧化氢诱导心肌细胞氧化损伤的保护作用[D]. 兰州. 兰州大学,2010.
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