【作者】 刘英；

【导师】 孙景辉；

【作者基本信息】 吉林大学 ， 内科学， 2007， 博士

【摘要】 本实验建立H9c2大鼠心肌细胞缺氧缺血模型,采用MTT法测定细胞存活率、流式细胞术测定细胞凋亡率、免疫细胞化学法检测Cyt C及Caspase-3蛋白表达,并应用FDP进行干预。结果表明:缺氧12小时后H9c2细胞存活率降低、凋亡率升高,Cyt C及Caspase-3蛋白表达增高;FDP提高细胞存活率,降低细胞凋亡率及Cyt C、Caspase-3蛋白表达。结扎7日龄新生大鼠一侧颈总动脉后,吸入低浓度氧复制HIBD模型,证实了它是一个稳定、可重复性好,设备简单,可大规模应用的模拟新生儿HIBD的动物模型。在此模型基础上,实验分为3组:HIBD组、FDP组及NC组。应用TUNEL检测法发现HIBD后12h心肌细胞凋亡明显增高,72h达高峰,7d仍有少量凋亡存在;FDP组较HIBD组心肌细胞凋亡率降低。应用免疫组化法,首次研究线粒体通道上的凋亡相关基因Caspase-3、Bax、Bcl-2及Cyt C在新生大鼠HIBD后不同时间点心肌细胞的表达,结果表明:Caspase-3、Bax、Bcl-2及Cyt C蛋白表达增高,且与凋亡细胞数之间呈直线正相关;FDP抑制Bax、Caspase-3、Cyt C蛋白表达,提高Bcl-2蛋白表达及Bcl-2/Bax的比值。同时动态观察HIBD后血清MDA、SOD及NO含量,结果表明:HIBD后血清MDA、NO增高,SOD降低,MDA与凋亡细胞数之间呈直线正相关,SOD与凋亡细胞数呈直线负相关;FDP可降低MDA,增高SOD。提示FDP可能通过抑制心肌细胞凋亡而发挥保护心肌细胞的作用。更多还原

【Abstract】 Neonatal hypoxic and ischemic encephalopathy (HIE) is a common disease in neonatal period and has high mortality and sequelae. Besides brain damage, it often causes functional disturbance of many organs. Heart is the most damaged organ. The usual reason of HIE is fetal distress in uterus and neonatal asphyxia. Asphyxia and hypoxia is pathogenesis of organ damage and functional disturbance. Until now, mechanism of heart injury about HIBD is unclear.Recently apoptosis has become the major research focus all over the world. Now it has been confirmed that cardiomyocyte apoptosis resides in many physiological and pathological change of cardiovascular disease. At present the report about neurocyte apoptosis on HIE are seen repeatedly home and abroad., but there are few research about cardiomyocyte apoptosis,and expression on apoptosis related gene, free radicle, effect of nitrogen monoxidum for cardiomyocyte apoptosis have not reported yet. Cardiac muscle is a kind of tissue needing high oxygen. More and more evidences show chondriosome has basic effects in cell apoptosis. Therefore, it is necessary for us to study the factor about affecting cell apoptosis in chondriosome pervium. This study is about cardiomyocyte apoptosis at hypoxic- ischema, influencing factors and protective effect of fructose 1,6-diphosphote (FDP) for cardiomyocyte.There are three parts in this study.1. H9c2 rat cardiomyocyte in vitro culture lack oxygen 12 hours, then make HI models of cardiomyocyte in vitro. We observed survival rate of cardiomyocyte by MTT means, H9c2 cardiomyocyte apoptosis rate by Annexin-V/PI double fluorescent labeling flow cytometry (FCM), expression of Cyt C and Caspase-3 protein by Immunocytochemistry. We set up FDP high dose intervention group, low dose intervention group and normal control group, and then observe protective effect for H9c2 cardiomyocyte apoptosis.2. At the base of vitro experiment ,this study prepared homoplastic HIBD animal models as human beings. We ligated one side common carotid artery of 7days rats, and reproduce HIBD model. For confirming its feasibility we use biochemistry rate to determine lactate dehydrogenase(LDH), isoenzyme of creatine kinase (CK) and blood gas analysis. At last common appearance, heart body, survival rate and weigh growth were observed.3. To identify the presence of cardiomyocyte apoptosis, the time of apoptosis, apoptosis relation genes (Cyt C, Caspase-3, Bax and Bcl-2) and the effect of free radicle and nitric oxide (NO) on cardiomyocyte apoptosis, all rats in the study were divided into sham operation normal control group(NC group), HI model group, FDP interventing group(1g/kg, ip, immediately, 24h, 48h, 72h after hypoxia every time, together 4 times). At time of 1h、4h、12h、24h、48h、72h、7d after HIBD, rats were put to death by decapitation and heart tissues were obtained. Heart tissue pathological change were observed by HE dyeing, cardiomyocyte apoptosis were observed by deoxyuridine triphosphate-biotin nick end labeling (TUNEL), and Bax、Bcl-2、Cyt C、Caspase-3 protein gene expression relation to apoptosis by immunohistochemistry. Simultaneously we determined malondialdehyde (MDA), erythrocuprein (SOD), oxidize cytokine NO by TBA chromatometry, xanthine oxidase method, nitrate reductase method at different time points. We also approach effect of free radical and NO for cardiomyocyte apoptosis, and then we study the effect of apoptosis relation gene, free radicle and NO on cardiomyocyte apoptosis.The results show:1. MTT method determines survival rate of cardiomyocyte: the survival rate of HI groups was lower than that of the control group (P<0.05). The survival rate of FDP group was higher than that of HI group, and FDP high dose group, difference is significant statistically (P<0.05).2. AnnexinV/PI (FCM) determines the change of apoptosis: Normal control group had little apoptosis, and apoptosis rates of HI groups were higher than that of normal control group. Apoptosis rates of FDP high dose group were lower than that of HI group, and difference is significant statistically (P<0.05). It showed that FDP may lead to resisting apoptosis.3. HI group in light microscope by HE dying had cell shrinkage and nucleus became smaller.4. Cyt C、Caspase-3 protein expression:There was weakly positive expression in cardiomyocyte kytoplasm of normal control group. Cyt C、Caspase-3 protein expression of HI group was obviously higher, but Cyt C、Caspase-3 protein expression of FDP group was lower than that of HI group, especially FDP high dose group (P<0.05). It showed that Cyt C、Caspase-3 can promote cardiomyocyte apoptosis and FDP can inhibit cardiomyocyte apoptosis by cutting down Cyt C、Caspase-3 protein expression.5. After preparation of HIBD,Models rats showed up different HI symptoms, and with increasement of day age, ,the weight of HIBD group grow slowly and was lagged than normal control group. Rats in HIBD group had the following symptoms:heart rate fastly, weak heart muscle contraction, irregular rhythm, dull cardiac muscle and venous blood color. HIBD group PaO2, PaSO2, HCO3- and pH were lower than those of the control group, and showed hypoxemia and metabolic acidosis. The survival rate of control group was 95%, but HIBD group was 75%. Serum cardiac creatase CK and LDH of HIBD group were obviously higher than those of the control group. Based on the appearance, heart body, blood gas analysis and cardiac creatase determition confirmed that this model was simple, easy, repeatable, and feasible, consistant with myocardial anoxia and ischemic models.6. HE dying: Cardiomyocyte of normal neonatal rats ranged regularly, edge sharpness, caryotheca and chromatospherite visible, but after HIBD cardiomyocyte ranged in disorder.7. Apoptosis of all neonatal rats at different time after HI: There were positive cells in NC group by chance, but cardiomyocyte nucleus was blue. After 12 hours apoptosis cells began increasing, and nucleus of TUNEL positive apoptosis cells were brown. Then they increased gradually, and at 72 hours it reach the peak. At 7 days apoptosis cells reduced, but were still higher than normal control group. Apoptosis cells reduced gradully and it was clear at the time of 48-72 hours. This confirmed that FDP could inhibit cardiomyocyte apoptosis and the effect was obvious after using three or four times.8. The results show that the expression of Cyt C in heart tissue is weakly positive in NC group. At the time of 12 hours, more positive cells appear in HIBD group. Cyt C was stained brown and appears to be trabs or particles in endochylema. Then the concentration of Cyt C multiplicated piecemeal, and the peak arrived at the time of 72 hours. Accompanied with the multiplition of Cyt C, apoptosis cells appear more. And it was suggested that the enhancement of Cyt C to apoptosis be possible in a further step. The expression of Caspase-3 in heart tissue of neonate rats was weakly positive in the normal contrast group. The color and distribution of Caspase-3 were the same as Cyt C. At the time of 12 hours, more positive cells appeared in HIBD group. Then the concentration of Caspase-3 multiplicated piecemeal, and it reached the peak at the time of 48 hours. The concentration appeared to be a slow descent. At the time of 7 days there was higher concentration than that of normal control group. With the multiplication of Caspase-3, the quantity of apoptosis cells became more. And it was also suggested that the enhancement of Caspase-3 to apoptosis be confirmed. The results of our experiment showed that the expression of Bcl-2 in heart tissue of neonate rats was weakly positive in the normal control groups. And it rised after HIBD. It reached the peak at the time of 48 hours and then descended. In contrast, the expression of Bax can be seen in the heart issue in NC group and the expression was noticeable after HIBD. The ratio of Bcl-2 and Bax reduced after HIBD. The result showed that Bax can activate the process of apoptosis and the effect of Bcl-2 was inverse. The process of cardiomyocyte apoptosis was depressed in different degree in FDP intervention group by the way of decreasing the expression of Cyt C and Caspase-3. The results of higher Bcl-2/Bax in FDP intervention group than that in HIBD group showed that the contribution of FDP had the business with Bcl-2 protein.9. The multiplication of MDA after HIBD began at the time of 4 hours, and it reached the peak at the time of 48 hours, then lowered down and appeared normal after 7days by observing the change of MDA、SOD、NO at different time points. The results indicated that lipoid peroxidation existed after HIBD. LPO emerged after the reaction of free radical and unsaturated fatty acid. The main metabolic product of LPO would promote the enhancement of MDA. And the multiplication of MDA had the positive correlation with cell apoptosis numbers.The concentration of SOD decreased at the time of 4 hours, and reach the lowest level at the time of 48h, and then recovered slowly. The multiplication of SOD had the negative correlation with cell apoptosis. This showed that multiplication of MDA and concentration of SOD loss may promote apoptosis emergence. Level of NO began at the time of 4 hours, and the peak reached at the time of 24 hours, then remained at higher level and appeared normal after 7 days. It showed that level of NO did not have the positive correlation with cell apoptosis, but had negative correlation with SOD and the positive correlation with MDA.Therefore, NO maybe promote cardiomyocyte apoptosis by indirect free radicle damage. In this experiment it was observed the change of MDA、SOD、NO in FDP group and effect after therapy 48-72 hours by FDP was better than HIBD group. It confirmed that FDP can resist oxygen free radical antioxidation.In summary, cardiomyocyte apoptosis after HIBD is very complicated, chondrosome may play an important role and the upper component element is oxidative stress mechanism of free radicle and NO. Expression of Bcl-2 gene on mitochondria membrane may be lower regulateing component element. Then by the mechanism of Cyt C release and Caspase-3 activation, cardiomyocyte apoptosis is induced.Regulation mechanism of cardiomyocyte apoptosis is very complicated, and it relates to many component element including influencing factors, signal passage and gene regulation.Many factors participate in every component element. This study observed the expression of Caspase-3, Bcl-2, Bax and Cyt C in plastiosome passage and MDA, SOD, NO. To some degree,there must be some limitations in explaining the mechanism of cardiomyocyte apoptosis of neonatal rats by HIBD. It should be studied deeply.更多还原