【作者】 马燕花；

【导师】 赵健雄； 李应东；

【作者基本信息】 兰州大学 ， 中西医结合内科临床， 2010， 博士

【摘要】 一、研究背景心血管疾病在我国的发病率逐年增加,目前已经成为造成我国人民死亡的首要疾病。近年来的大量研究表明,心肌细胞凋亡是多种心血管疾病如心力衰竭、扩张性心肌病(DCM)、阿霉素诱导性心肌病和病毒性心肌炎、心肌梗塞、心肌缺血等的一个重要的发生和进展机制。同时细胞凋亡参与心室重构和心功能不全的发生,是心功能远期预后的重要预报因子。缺氧、缺氧复氧、酸中毒、氧化应激、压力应激等多种刺激均可诱导心肌细胞产生凋亡。其中,氧自由基代谢障碍是心肌细胞凋亡的重要促发因素,生物体内存在的活性氧可以与DNA、蛋白质、多元不饱和脂肪酸等作用,造成DNA链断裂、突变和对热稳定性的改变,严重影响遗传信息的传递,使细胞代谢功能紊乱；导致蛋白质与蛋白质交联、蛋白质与DNA交联,使其理化性质和生物学功能发生改变；作用于生物膜的多不饱和脂肪酸,使其发生脂质过氧化,造成膜的完整性被破坏、膜流动性下降、膜脆性增加,影响细胞内外物质与信息的交换。当活性氧过氧化氢(H2O2)高浓度作用于心肌细胞时,可致细胞内活性氧水平迅速增加,并诱导体内抗氧化剂如：超氧化物歧化酶、过氧化氢酶、谷胱苷肽过氧化物酶等的活性降低,使生物膜的结构和功能的完整性遭到破坏,致使细胞发生凋亡。细胞凋亡又称程序性死亡(PCD),是多细胞有机体为保持自身组织稳定、调控自身细胞的增殖和死亡之间的平衡、由基因控制的细胞主动性死亡过程。诱导细胞凋亡的因素很多,体内代谢或外源性因素产生的自由基均被证实可诱导细胞凋亡。在细胞凋亡的发生机制研究中,已知的引起细胞凋亡的信号传导通路有：死亡因子介导的细胞凋亡；神经酰胺介导的细胞凋亡；p53+基因触发的细胞凋亡；线粒体损伤启动的细胞凋亡。通过以上通路,细胞凋亡的结果使体细胞特别是具有重要功能的细胞如脑细胞、心肌细胞数量减少,造成其组成的重要器官如心室肌等发生衰退性病理过程。在心肌细胞凋亡中研究较多的有两条主要的信号转导通路,即线粒体通路和膜死亡受体通路。通过干预心肌细胞凋亡能在一定程度上保护心肌细胞,因此,人们在研究药物对心肌细胞凋亡影响方面产生了浓厚的兴趣。长期以来,中药在心血管疾病的治疗中占据了一定的地位。目前,中药与心肌细胞凋亡相关性的研究正受到普遍关注,并取得了一些可喜的成果,其研究正在不断深入,尤其在探索中药有效单体成分对心肌细胞凋亡的影响方面。本实验室运用超滤膜技术对传统方剂当归补血汤进行分离纯化,前期研究已证实,当归红芪超滤膜提取物具有清除氧自由基,抗脂质过氧化,抗心肌细胞凋亡的作用。在本课题中,通过模拟体内心肌细胞氧化损伤过程,建立H2O2诱导体外培养心肌细胞凋亡模型,观察当归红芪超滤膜提取物对氧化损伤诱导的体外培养心肌细胞凋亡的影响,从而为明确当归红芪超滤膜提取物抗凋亡的作用机制及其临床应用价值奠定基础。二、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。三、方法1、应用陶瓷膜微滤和聚丙烯腈(PAN)中空纤维超滤膜(截留分子量为10万分子量)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：取出生后1～3d的大鼠乳鼠,用75%乙醇溶液浸泡消毒皮肤后,移入超净工作台。开胸取出心室肌,运用酶消化法反复多次消化,收集细胞悬液,以差速贴壁法分离纯化心肌细胞,用含15%小牛血清的DF培养液调整细胞浓度至每毫升5×105,在37℃、5% CO2条件下培养。选生长状态佳者于72 h后进行实验。采用台盼蓝染色法检测心肌细胞存活率。采用心肌细胞α横纹肌肌动蛋白(a-Sarcomericactin)的免疫细胞化学染色方法鉴定心肌细胞纯度。3、制备乳鼠心肌细胞氧化应激性损伤模型：以400μmol·L-1的H2O2诱导心肌细胞凋亡。实验分为以下5组：正常对照组；H202模型组(作用6h)；当归红芪超滤膜提取物高剂量干预组(15 g·L-1)、当归红芪超滤膜提取物中剂量(7.5 g·L-1)干预组、当归红芪超滤膜提取物低剂量(3.75·g-1)干预组。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。四、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(不含阿魏酸)多糖含量接近单剂含量,当归多糖含量为23.84 g·L-1,红芪多糖含量为27.55 g·L-1,合剂多糖含量为22.08g·L-1。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、低剂量干预组心肌细胞搏动频率分别为86.9±8.4、71.1±6.7和43.7±9.6次／min,细胞伪足较对照组变细,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严重受损,细胞存活率显著降低(29.5%),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(274.36±15.16U/L)、CK(201.47±2.71 U/L)的含量增加,与正常对照组比较(LDH 138.07±11.27U/L,CK42.69±1.53 U/L),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(LDH 167.22±10.11U/L, CK 88.14±1.06U/L)、中剂量组(LDH183.48±9.88U/L, CK 99.66±4.18 U/L)、低剂量组(LDH201.06±18.13U/L, CK 132.54±1.92 U/L)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 (3.82±0.64nmol/mL)、MPO (114.00±3.24 U/L)含量增加、SOD (58.23±2.45U/mL)活力降低,与正常对照组(MDA2.34±0.50 nmol/mL, MPO 38.47±1.12 U/L, SOD 118.33±5.77 U/mL)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(MDA2.51±0.28 nmol/mL, MPO 59.76±1.84 U/L, SOD 101.34±2.14U/mL)、中剂量组(MDA 2.80±0.32 nmol/mL, MPO 71.52±1.03 U/L, SOD 83.36±6.63U/mL)、低剂量组(MDA 3.11±0.35 nmol/mL, MPO 85.22±4.18 U/L, SOD71.45±7.11U/mL)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蛋白的表达明显降低,与正常对照组比较差异具有显著性(P<0.01), Bcl-2/Bax的蛋白比值降低(P＜0.01)。而经当归红芪超滤膜提取物处理后,心肌细胞中Hsp70蛋白表达量均较模型组显著增加(P＜0.05)；在高剂量组和中剂量组细胞中Bcl-2蛋白表达显著增加(P<0.05), Bax蛋白表达显著降低(P＜0.01),Bcl-2/Bax比值显著升高(P＜0.01),差异具有统计学意义。低剂量组则Bcl-2、Bax蛋白表达无显著改变(P＞0.05)。五、结论1、氧化损伤是诱导心肌细胞凋亡的重要原因之一。2、当归红芪超滤膜提取物具有抗过氧化氢诱导心肌细胞损伤的作用,可减轻细胞活力的下降,抑制心肌细胞凋亡。3、Bax、Caspase-3的激活是过氧化氢诱导心肌细胞凋亡的重要机制,Bcl-2、Hsp70表达的增多具有抗心肌细胞凋亡作用。4、当归红芪超滤膜提取物可能通过抑制心肌细胞凋亡、防止心肌细胞氧化损伤而在治疗心血管病方面发挥作用。更多还原

【Abstract】 BackgroundCardiovascular disease is currently responsible for the death of the primary disease of our people, and in recent years, a large number of research evidence shows that cardiomyocyte apoptosis is an important mechanism for the occurrence and progress of heart failure, dilated cardiomyopathy (DCM), adriamycin-induced cardiomyopathy and viral myocarditis, myocardial infarction, myocardial ischemia and other cardiovascular diseases. Apoptosis is also involved in ventricular remodeling, and is a predictor of long-term prognosis of cardiac function.A variety of stimulus factors can induce apoptosis in cardiac cells, such as hypoxia, hypoxia-reoxygenation, acidosis, oxidative stress, pressure, stress, etc. Among them, Oxygen free radical metabolism disorder is an important precipitating factor in cardiomyocyte apoptosis. In vivo reactive oxygen species can with the role of DNA, proteins and polyunsaturated fatty acids Etc, and cause DNA strand breaks and oxidative damage, protein-protein cross-linking, protein-DNA cross-linking and lipid peroxidation. When reactive oxygen species H2O2 of high concentrations acts on cardiomyocyte, intracellular reactive oxygen species levels can be induced increase rapidly and in vivo the activity of anti-oxidants such as:superoxide dismutase, catalase, glutathione peroxidase enzymes can be induced decrease, so that the structure and function of membrane integrity have been destroyed and cell apoptosis has been caused.Cell apoptosis is an active death process controlled by gene, in which multi-cell organism maintain their autologous tissue and keep the balance between cell proliferation and death. In apoptosis studies, the known signal transduction pathways of apoptosis are:the death factor-mediated apoptosis; ceramide-mediated apoptosis; p53+gene-triggered apoptosis; mitochondrial damage-mediated apoptosis. In those many motivations, free radical produced in body metabolism or extrinsic factor can induce cell apoptosis. Through the above pathway, the results in cell apoptosis make the number of somatic cells reduce, especially those with important functions such as brain cells, myocardial cells, causing the vital organs such as the ventricular muscle a recession in pathological processes. In research of apoptosis in myocardial cells, the two main signal transduction pathways are proposed, namely mitochondrial pathway and the membrane death receptor pathway.It has been confirmed that inhibiting cadiomyocytes apoptosis can protect heart on certain degree. Scientists show great interests on the effect of drugs anti-apoptosis on cardiomyocytes. Ever since a long time ago, China medicine plays an important role in cardiovascular diseases treatment. Scientists are paying attention to the relation between China medicine and cardiomyocytes apoptosis, and have obtained some success. The researches have to be done further, especially on the effective simple component of China medicine.In our laboratory Dangguibuxuetang was isolated and purified by using ultrafiltration membrane technology on the traditional recipe. And preliminary studies have confirmed that the ultra-filtration extract from the mixture of angelica sinensis and hedysarum polybotrys (UFE-AH) has effect of clear oxygen free radicals, anti-lipid peroxidation and anti-apoptosis of myocardial cells. In this study we established the model of myocardial cell apoptosis H2O2-induced in vitro through simulating the process of oxidative damage of myocardial cells in vivo, and observed the effect of UFE-AH on apoptosis induced by oxidative stress in neonatal rat cardiomyocytes in vitro culture. It can identify the mechanism of UFE-AH’s anti-apoptosis effect and its value of clinical appliancation.Objective1.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.Methods1. Refined the mixture of angelica sinensis and hedysarum polybotrys (1:5) using ceramic membrane and PAN (Ultra-filtration membrane) of the hollow fiber (cut to stay the molecular weight to measure for 10,0000).2. Primary culture of neonatal rat cardiomyocytes:neonatal rats of 1-3 days were removed, soaked in disinfecting the skin with 75%ethanol, and moved to Clean Bench. Ventricular muscle was removed from chest and digested over and over again using enzymatic digestion. The cell suspension was collected and cardiomyocytes were Separated and Purified with differential adhesion method. Cardiomyocytes were cultivated under the conditions of 37℃,5%CO2, after the concentration of which had been adjusted to per ml of 5×105with DF medium of containing 15%fetal calf serum.Those with good growth state were elected to carry our experiments in the 72h. We detected the survival rate of cardiac cells using trypan blue staining. We identified myocardial cell purity with a-Sarcomericactin immunohistochemistry staining.3. Constructing oxidative stress mode by using peroxide:Myocardial cell apoptosis was induced by 400μmol-L-1 H2O2.There were 5 groups in our research:normal control group; model group(pretreatment myocardial cells with H2O2 for 6h); UFE-AH high-dose intervention group(pretreatment myocardial cells with UFE-AH of 15 g-L"1 for 48h, H); UFE-AH middle-dose intervention group(pretreatment myocardial cells with UFE-AH of 7.5 g-L"1 for 48h, M); UFE-AH low-dose intervention group(pretreatment myocardial cells with UFE-AH of 3.75 g-L-1 for 48h, L). The morphological changes of myocardial cell were observed under inverted phase contrast microscope; The cell viability was detected by MTT chromatometry; Myocardial cell apoptosis was detected by Annexin V-FITC/PI double staining marks; Myocardial cell apoptosis index was detected by flow cytometry in each group.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, MDAand MPO.RESULETS1. 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 (Angelica polysaccharide content of 23.84 g-L"1, HPS content of 27.55 g-L"1, mixture content of 22.08 g-L"1). 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 survival rates were 77.6%, 64.4%,43.9, and were significantly increased compared with oxidative injury model group (P< 0.01).4. Biochemistry test:The permeability of cell membrane was increased by H2O2 treatment. So the levels of LDH(274.36±15.16U/L) and CK(201.47±2.71U/L) in the culture medium were increased, and compared with the control group (LDH 138.07±11.27U/L, CK 42.69±1.53U/L), the content of LDH and CK were significant differences (P<0.01). UFE-AH could significantly inhibit the changes of myocardial cell membrane permeability H2O2-induced and reduce the leakage of LDH and CK.The leakage of LDH and CK in high-dose group (LDH 167.22±10.11 U/L, CK 88.14±1.06U/L), middle-dose group (LDH 183.48±9.88U/L, CK 99.66±4.18U/L) and low-dose group (LDH 201.06±18.13U/L, CK 132.54±1.92U/L) were significantly reduced, and there were significantly different compared with the model group (P<0.01).The contents of MDA(3.82±0.64nmol/mL)and MPO(114.00±3.24U/L)were increased and the activity of SOD (58.23±2.45U/mL) was reduced in H2O2 injury group. UFE-AH could significantly reduce the intracellular contents of MDA(H:2.51±0.28nmol/mL; M:2.80±0.32nmol/mL; L:3.11±0.35 nmol/ mL) and MPO(H:59.76±1.84U/L; M:71.52±1.03U/L; L:85.22±4.18U/L), and increase the activity of SOD (H:101.34±2.14U/mL; M:83.36±6.63U/mL; L:71.45±7.11U/mL). The difference was significant between H2O2 injury group and UFE-AH treatment group (P<0.01). And dose-dependent manner, the contents of LDH, CK, MDA and MPO were decreased and the activity of SOD was increased gradually with the concentration of UFE-AH increasing.5. RT-PCR test results:The mRNA of Hsp70, Caspase-3, Bcl-2 and Bax showed low expression in normal control group.In model group, the expression of Hsp70, Caspase-3, and Bax mRNA were increased significantly and the expression of Bcl-2 mRNA was significantly reduced. The difference between model group and normal control group was statistically significant (P< 0.05).6.Western-blotting test results:The expression of Hsp70 and Bax protein were significantly increased and Bcl-2 protein was significantly reduced in myocardial cells after having been treated for 6h by 400μmol·L-1 H2O2. There were significant difference compared with the normal control group (P<0.01). And the protein ratio of Bcl-2/Bax was decreased (P<0.01).However, in myocardial cells after UFE-AH treatment, the expression of Hsp70 protein was increased significantly than those in model group (P<0.05); In myocardial cells of high and middle dose group, the expression of Bcl-2 protein was increased significantly (P<0.05), Bax was markedly reduced (P<0.01), and the ratio of Bcl-2/Bax was increased significantly (P<0.01); There was statistical significance. But in low-dose group, the expression of Bcl-2 and Bax protein had not changed significantly (P>0.05).Conclusions1. Oxidative stress is a major reason of inducing cardiomyocyte apoptosis.2.UFE-AH has an effect of anti anti-oxidative stress of myocardialcells induced by H2O2, It can reduce the decline of cell viability and inhibit myocardial cell apoptosis.3.The activation of Bax and Caspase-3 plays an important role in myocardial cell apoptosis, and the increases of Bcl-2 and Hsp70 expression have an anti-myocardial cell apoptosis effect.4. UFE-AH may play a role in the treatment of cardiovascular disease by inhibiting apoptosis of myocardial cells and preventing oxidative damage of myocardial cells.更多还原