阿霉素致大鼠心肌线粒体DNA突变及硒的保护作用

采用ＰＣＲ方法对大鼠心肌ｍｔＤＮＡ特异片段进行扩增，用ＰＣＲ－ＳＳＣＰ和薄层扫描方法检测其片段的缺失突变和点突变。阿霉素组大鼠心肌ｍｔＤＮＡ特异片段出现缺失突变（６０．５％），并有点突变（２／６）。Ｓｅ保护组检出部分缺失突变（３０．０％），未检出点突变。阿霉素可以导致大鼠心肌ｍｔＤＮＡ突变热点区域发生缺失突变和点突变。ｍｔＤＮＡ损伤可能是阿霉素致心肌损伤的重要机制之一。Ｓｅ可以降低阿霉素对ｍｔＤＮＡ的损伤，对阿霉素性心肌损伤有明显地保护作用。     

心肌和淋巴细胞线粒体DNA缺失在衰老中的意义

用聚合酶链反应（ＰＣＲ）技术对５０例心脏病患者及健康人线粒体ＤＮＡ（ｍｔＤＮＡ）中遗传物质缺失的研究发现，随年龄增长，发生缺失的ｍｔＤＮＡ比例增加。淋巴细胞和心肌细胞ｍｔＤＮＡ均存在包含有７．４３６ｋｂ缺失的片断，而前者有多重缺失，发生缺失的ｍｔＤＮＡ片断约占正常ｍｔＤＮＡ的１０．３％。提示ｍｔＤＮＡ的缺失可能在衰老和老年人心脏病的发生机制中有一定意义。     

心力衰竭患者心肌线粒体DNA缺失突变的定量分析及与心功能损害程度的关系

目的为探讨心肌线粒体ＤＮＡ（ｍｔＤＮＡ）获得性损伤在衰竭心肌发病中的意义。方法对２８例慢性心力衰竭患者活检心肌，用定量ＰＣＲ方法，以常发缺失型突变ｍｔＤＮＡ４９７７ｂｐ（ｍｔ－ＤＮＡ４９７７）和７４３６ｂｐ（ｍｔＤＮＡ７４３６）缺失率为指标，观察心力衰竭不同发展阶段ｍｔＤＮＡ损伤程度与患者临床心功能受损之间的关系。结果慢性心力衰竭患者活检心肌中ｍｔＤＮＡ４９７７缺失频率为１００％，缺失率在０．１０３％～１．０２８％之间；７０％的扩张型心肌病（ＤＣＭ）和３８．９％的风湿性心脏病（ＲＨＤ）患者存在ｍｔＤＮＡ７４３６缺失，缺失率在０．００９％～０．４８８％之间。１０例健康对照者中，仅２例４５岁以上者有低水平的ｍｔＤＮＡ缺失。心功能愈差，左房扩大愈明显者，ｍｔＤＮＡ缺失率愈高；扩张型心肌病缺失程度明显高于风湿性心脏病。结论心肌ｍｔＤＮＡ损伤与心脏功能的受损程度密切相关，特别是ＤＣＭ患者。     

衰老中线粒体脱氧核糖核酸的突变

一、突变机制　　与衰老相关线粒体脱氧核糖核酸（ｍｉｔｏｃｈｏｎｄｒｉａｌＤＮＡ，ｍｔＤＮＡ）的突变有两种：缺失及点突变〔１〕。研究较多的是ｍｔＤＮＡ大片段缺失，对其产生的机制有如下几种观点：　　１．经由同向重复序列两侧基因重组可能引起ｍｔＤＮＡ大片段缺失。在研究人及动物ｍｔＤＮＡ缺失时发现，多数缺失位点的两侧存在着长度不一的同向重复序列，有两种形式，一是缺失点的两侧有精确的同向重复序列，５’末端紧位于缺失点左侧，而另一端的重复位于３’末端缺失区内，紧邻右侧的缺失点，在人类同向重复序列为５～１３ｂ…     

老年鼠肝,脑细胞核活性基因的变化

我室用ＤＮＡ酶Ⅰ为活性基因探针酶解ＤＮＡ，以ＤＮＡ酶Ⅱ酶解ＤＮＡ为对照，研究老年鼠肝、脑细胞核活性基因的变化。发现在不同的消化时间内，老年鼠肝、脑细胞核对ＤＮＡ酶Ⅰ消化敏感性小于青年鼠，尤以消化２分钟为明显，说明老年鼠肝、脑细胞核内活性基因数在减少。     

线粒体DNA点突变与心肌病关系的研究

用聚合酶链反应（ＰＣＲ）和异源双链（ＨＥＴ）凝胶电泳检测了１５例原发性扩张型心肌病（ＤＣＭ）、１３例急性心肌炎患儿及１个肥厚型心肌病（ＨＣＭ）家系中１７例成员 的外周血线粒体ＤＮＡ（ｍｔＤＮＡ）点突变。结果显示，ＤＣＭ患儿中，６例在ｍｔＤＮＡ保守区３１０８￣３７１７位存在点突变，其中１例家族性ＤＣＭ患儿及其母亲均检出点突变，提示ｍｔＤＮＡ点突变在ＤＣＭ发病中起一定作用；１３例急性心肌炎患儿中有１例     

衰老小鼠线粒体DNA缺失的研究

目的：探讨线粒体DNA(mtDNA)大片段缺失与衰老的关系。方法：用聚合酶链反应（PCR）检测不同年龄段小鼠心肌组织mtDNA片段缺失，同时用电镜观察其组织学改变。结果：用PCR检测到2只老年小鼠心肌mtDNA存在着3.87Kb片段缺失，电镜下老年小鼠心肌线粒体明显肥大肿胀，数目减少。结论：MtDNA片段缺失的发生随年龄增加而增加，可能中衰老的过程中起重要作用。     

心力衰竭患者心肌线粒体DNA缺失突变的定量分析及与心功能？…

为探讨心肌线粒体ＤＮＡ获得性损伤在衰竭心肌发病中的意义。方法对２８例慢性心力衰竭患者活检心肌，用定量ＰＣＲ方法，以常发缺失型突ｍｔＤＮＡ４９７７ｂｐ和７４３６ｂｐ缺失率为指标，观察心力衰竭不同发展阶段ｍｔＤＮＡ损伤程度与患者临床心功能受损之间的关系。     

聚合酶链反应检测恶性疟原虫的研究

根据编码恶性疟原虫红细胞结合抗原（ＥＢＡ－１７５）的部分ＤＮＡ片段而设计合成寡核苷酸引物并进行多聚酶链反应（ＰＣＲ）以检测体外培养的恶性疟原虫（Ｐ．ｆ．）。扩增产物经琼脂糖凝胶电泳分析，可见扩增出了特异的４９２ｂｐ大小的ＤＮＡ片段，而对间日疟原虫（Ｐ．ｖ．）、食蟹猴疟原虫（Ｐ．ｃ．）、约氏鼠疟原虫（Ｐ．ｙ．）、伯氏鼠疟原虫（Ｐ．ｂ．）及正常人血白细胞ＤＮＡ不能扩增出此片段。本法可检原虫密度下限为２０μｌ血中仅含１０个原虫，具有高敏感性和特异性。     

初步筛选心血管相关基因——表达序列片段测定法

目的从正常人胎儿心脏ｃＤＮＡ文库中，用ＤＮＡ自动测序的方法筛选心血管相关基因。方法（１）人胚胎心ｃＤＮＡ文库的构建；（２）挑取有目的基因片段插入的噬菌斑，选用正向Ｔ３和反向Ｔ７引物做ＰＣＲ扩增；（３）直接使用其ＰＣＲ产物做ＤＮＡ测序模板，用荧光标记的Ｔ３Ｂ引物做再次ＰＣＲ扩增；（４）用ＤＮＡ测序仪（ｐｈａｒｍａｃｉａ）测定基因表达序列片段（ＥＳＴｓ）；（５）将所获得的ＥＳＴｓ输入Ｇｅｎｅｂａｎｋ数据库做同源序列分析。结果（１）所构建的人胎儿心脏ｃＤＮＡ文库的库容量为１×１０９克隆，平均目的基因片段１．２～１．５ｋｂ，ＰＣＲ产物阳性克隆检出率高达７０％以上，用６％尿素－聚丙烯酰胺凝胶电泳５ｈ，可测ｃＤＮＡ序列片段２１０～５５０ｂｐ，平均３００ｂｐ；（２）在所测３１３２个克隆的ＥＳＴｓ中，新ＥＳＴｓ为４７．４％，已知序列片段为４４．１％。结论利用人胎儿心脏ｃＤＮＡ文库测定ＥＳＴｓ是筛选心血管基因的有效方法；构建高质量的ｃＤＮＡ文库是成功测定ＥＳＴｓ的保证。     

The effects of idebenone on DNA and RNA contents as well as synthesis rates of total and poly(A)+ RNA in brain of normal, old C57BL/6J mice and in experimental partial cerebral ischemia of rats

Effects of idebenone on RNA and DNA contents as well as on synthesis rates of total and poly(A)(+) RNA in the brain were measured in two animal models: (1) Normal young and old, male C57BL/6J mice (6 and 32 months). Idebenone suspended in 5% gum arabic was applied in 50 mg/kg/day dose to old mice for 1 month through a gastric tube. (2) Adult female CFY rats (14-18 months) in which experimental partial cerebral ischemia was induced by bilateral common carotid artery occlusion. Idebenone was administered intraperitoneally in two dose (10mg/kg and 100 mg/kg body weight) 30 min before the interruption of carotid blood flow. DNA content remained invariate during aging in the brain; idebenone treatment did not exert any influence on this parameter. RNA content as well as total and poly(A)(+) RNA synthesis rates, which were measured by incorporation of tritiated uridine into RNA, decreased significantly with age in brain. Idebenone treatment did not cause any essential change of the metabolism of RNA under the given conditions. The RNA and DNA contents of brain were influenced neither by experimental partial cerebral ischemia nor by treatment with idebenone during the ischemia. Partial cerebral ischemia decreased the rate of total and poly(A)(+) RNA synthesis in the brain about 15-45% depending on the methods and basis of expression. This decline could totally be prevented by intraperitoneal application of 10 mg/kg idebenone 30 min before the onset of the partial ischemia. The dose of 100 mg/kg idebenone also elevated the rate of RNA synthesis; however, this increase remained statistically insignificant.     

病毒性心肌炎小鼠心肌与骨骼肌细胞线粒体损伤及其相关性

目的探讨病毒性心肌炎（VMC）小鼠心肌与骨骼肌细胞线粒体损伤（线粒体膜磷脂脱失和线粒体DNA3867缺失）程度及二者的相关性。方法50只BALB/c小鼠随机分为2组,实验组（40只）腹腔注射内含柯萨奇B3病毒（Coxsackievirus B3,CVB3,TCID50=108）的Eagle液制备VMC小鼠模型,另10只为对照组。分别于病毒感染后3、11和24 d行心肌和骨骼肌细胞线粒体膜磷脂脱失程度和mtDNA3867缺失率的测定,并用Spearman法对其进行相关分析。结果实验组小鼠在病毒感染后3 d,可见心肌和骨骼肌细胞mtDNA3867缺失率显著高于对照组（P<0.05）,而线粒体膜磷脂脱失程度与对照组相比无显著性差异;病毒感染后11 d,心肌和骨骼肌细胞mtDNA3867缺失性损伤达高峰（P<0.05）,线粒体膜磷脂脱失程度亦显著高于对照组（P<0.05）;病毒感染后24 d,心肌和骨骼肌细胞线粒体膜磷脂脱失和mtDNA3867缺失程度与感染后11 d组比较无显著性差异,但与对照组和病毒感染后3 d组比较,仍有显著性差异（P<0.05）。线粒体的上述损伤性改变在心肌和骨骼肌细胞呈一致性同步变化,且具有良好相关性（P<0.05）。结论CVB3可显著损伤心肌和骨骼肌线粒体DNA和膜磷脂,两者损伤具有相关性,提示骨骼肌有望成为反映心肌细胞线粒体损伤的外周细胞“窗口”。     

增龄对SAM小鼠脑线粒体DNA氧化损伤的影响

目的探讨增龄对SAM小鼠脑线粒体DNA(mtDNA)缺失、活性氧自由基水平及脑组织总抗氧化能力的影响。方法SAM-P／8和SAM-R／1小鼠分别分为三组幼年组(0．5月龄)、青年组(2月龄)和老年组(12月龄)各6只。用PCR方法测定脑组织线粒体DNA缺失、用化学发光法测定总抗氧化能力及活性氧自由基。结果随增龄，SAM—P／8小鼠脑组织线粒体DNA缺失增加，线粒体活性氧自由基水平升高，脑组织总抗氧化能力下降。结论随增龄，SAM-P／8小鼠脑组织氧化损伤增加，可能是导致其记忆学习障碍的原因之一。     

Differential expression of various cytokine receptors in the brain after stimulation with LPS in young and old mice.

The effect of lipopolysaccharides (LPS) on the expression of cytokine receptors was examined in the spleen, brain and pituitary gland, and compared in young and old mice. The level of mRNA for various cytokine receptors (IL-1RI, IL-2Ralpha, IL-3Ralpha, IL-6R, TNFalphaR and IFNgammaR) was found to be increased in the spleen of young but not in old mice within 2-6h of stimulation with LPS. Similar enhancement of cytokine receptor mRNA was also observed in the brain after LPS stimulation, but the magnitude varied according to the type of cytokine receptor, the site of brain and the age of the mice.In the hypothalamus, the level of mRNA for IL-1R, IL-3R, IL-6R and IFNgammaR increased in young but not in old mice. Reciprocally, in the cerebral cortex, mRNA for TNFalphaR and IFNgammaR increased in old but not in young mice. In the hippocampus, TNFalphaR mRNA expression, increased in young but not in old mice, and expression of the other cytokine receptors did not change greatly in either. In the pituitary gland, mRNA for IL-6R, TNFalphaR and IFNgammaR increased in both young and old mice, but IL-2Ralpha increased only in young mice.Thus, various cytokines produced by immune cells might directly or indirectly influence brain functions through the various cytokine receptors expressed in the brain. Moreover, interactions between the immune system and the brain at the time of infection would be expected to be different in young and old mice, because cytokine production changes with age, as does the expression of their receptors in the brain.     

鸡胚低分子提取物对D-半乳糖拟衰老小鼠线粒体DNA缺失突变的影响

目的 探讨鸡胚低分子提取物对D-半乳糖诱导的衰老小鼠线粒体DNA(mtDNA)缺失突变的影响.方法 采用D-半乳糖诱导制备衰老小鼠模型并用鸡胚低分子提取物处理.用聚合酶链反应技术和琼脂糖凝胶电泳检测mtDNA缺失片段,密度扫描技术对扩增片段进行相对定量.缺失片段构建质粒后,直接测序进行鉴定.结果 全部小鼠的肝、大脑皮质与海马组织内均存在4239 bp的mtDNA缺失片段.衰老模型鼠不同组织mtDNA缺失的相对百分含量均明显高于正常对照组(均为P%0.01),而鸡胚低分子提取物可明显降低模型鼠肝、大脑皮质与海马组织内mtDNA缺失的比例(均为P＜0.05).肝组织中mtDNA缺失的比例较大脑皮质和海马组织更高.结论 4239 bp的mtDNA缺失片段普遍存在于小鼠中,鸡胚的低分子提取物可以明显减少D-半乳糖拟衰老小鼠4239 bp的mtDNA缺失的发生.     

Age-dependent accumulation of mitochondrial DNA deletions in the aortic root of atherosclerosis-prone apolipoprotein E-knockout mice

PurposeTo investigate whether mitochondrial DNA (mtDNA) damage, specifically deletion, contributes to the development of atherosclerosis or is simply a secondary effect of the primary factors causing atherosclerosis.Materials and methodsmtDNA deletion was detected by PCR in the aortic root of atherosclerosis-prone C57BL/6J apolipoprotein (Apo) E gene deficient (−/−) mice and control C57BL/6J mice at different ages. Atherosclerotic plaques in the Apo E−/− mice were assessed using frozen sections of the aortic root. The protein levels of COX III and 8-oxoguanine glycosylase (OGG1) were determined.Resultswhile mtDNA deletions accumulated significantly in mice as young as 2- month-old, atherosclerotic plaques were not detected until mice were 6 months old or older, suggesting that mtDNA deletion occurs prior to the formation of atherosclerotic plaques in the aortic root of these mice. Moreover, the expression levels of mtDNA-encoded COX III protein in both 2-month-old and 16-month-old C57BL/6J ApoE−/− mice were significantly lower than those in C57BL/6J mice (p < 0.05). Additionally, the protein level of 8-oxoguanine glycosylase (OGG1), a mitochondrial enzyme that functions in DNA excision repair, decreased with age in these mice, indicating that age-related down-regulation of mtDNA excision repair also contributes to atherosclerosis in C57BL/6J ApoE−/− mice.ConclusionThese results reveal that mtDNA deletions occur during the early “initiation” stage of atherosclerosis in C57BL/6J ApoE−/− mice and have the potential to promote atherosclerosis.     

Effect of age in rats following middle cerebral artery occlusion

BACKGROUND: Despite the impressive increased understanding of the ischemic brain damage in general, the study of effects on different age groups, young versus old, using comparable ischemic insults is clearly lacking. OBJECTIVES: To investigate the mortality rate and neurological outcome among young and old rats, through a model of cerebral ischemia by middle cerebral artery occlusion (MCAO). METHODS: Twenty-three old (22-24 months of age) and 16 young (3-4 months of age) male rats underwent a MCAO procedure for 60 min. Surviving rats were randomly assigned to the 24-hour or 28-day resting group. The mortality rate and neurological outcome in different recovery time periods were determined for comparison between the young and old rats. RESULTS: The overall mortality rate in old rats (43.5%) was significantly higher than that of the young rats (6.3%) (p = 0.01). The infarct volume for the 24-hour post-MCAO was 181.86 +/- 11.87 mm(3) for the young rats, and 204.64 +/- 27.18 mm(3) for the old rats. For the 28-day post-MCAO, the value was 91.16 +/- 3.59 mm(3) for the young rats, and 103.38 +/- 26.43 mm(3) for the old rats. A significant reduction in infarct volume is noted in both young (p < 0.01) and old (p < 0.05) rats after 28 days of recovery compared to that after 24 h of recovery. There was no meaningful difference in infarct volume between the young and old rats measured at 24 h or 28 days after the ischemic procedure. The right volume was larger than the left volume at 24 h post-MCAO for both the young and the old rats, whereas the quotient approached unity for the young rats at 28 days post-MCAO. For the old rats, the quotient was negative at 28 days post-MCAO, representing the ipsilateral hemisphere was smaller than the contralateral hemisphere. CONCLUSION: The mortality risk to ischemic damage is greater for old rats. If an old rat survives the high-risk mortality in a short period after the MCAO procedure, the recovery would be of no difference to that of a young rat.     

Age-related differences in experimental stroke: possible involvement of mitochondrial dysfunction and oxidative damage

Age is the single most important risk factor for cerebral stroke. Unfortunately, the effect of age on ischemic brain damage is less clear. In this study, we sought to examine the potential influence of aging on the histologic and functional outcomes after ischemia. Juvenile (4 weeks of age), young adult (4 months of age), mid-aged (11-12 months of age), and aged (18-19 months of age) mice were subjected to transient middle cerebral artery occlusion. There was no remarkable difference of infarct volume on postoperative days 1 and 3. However, on postoperative day 7, aged mice exhibited significantly worsened infarct volume compared with juvenile and young mice. Intriguingly, the increase of infarct volume was most prominent in the striatal area rather than in cortex. Accordingly, aged mice displayed a slower and incomplete functional recovery after stroke. We further evaluated the effects of aging on the oxidative damage and mitochondrial dysfunction following ischemia. Brain tissues were assayed for lipid, DNA, and protein peroxidation products, mitochondrial enzyme activities, mitochondrial membrane potential, production of reactive oxygen species, and antioxidant activities. Aging was associated with declined mitochondrial function and antioxidant detoxification following ischemia, thereby inducing a deteriorated oxidative damage. Regional subanalyses demonstrated that, in accordance with infarct area, the pro-oxidant/antioxidant imbalance occurred more prominently in subcortical areas. Collectively, these findings suggest mitochondria-mediated oxidative damage may be involved in the age-related aggravated injury in subcortical areas. Mitochondrial protection could be a promising target for neuroprotective therapy, especially in the aged population.     

Carotid artery ligation-induced cerebral ischemia in old,male, Sprague-Dawleyrats

Virgin, male, Sprague-Dawley rats were maintained free of disease until they were 34 months old. These rats were subjected to acute cerebral ischemia by surgical ligation of one carotid artery; young (6 to 7 month-old) males were used for purposes of comparison. The old animals convulsed and died, all within a few hours of ligation, despite the protective effect of deep anesthesia. The young rats manifested extensor rigidity and other signs of cerebral ischemia but only 20% died. Serum iipids, glucose, corticosterone, and BUN levels were acutely elevated in the old but not the young rats concomitant with the onset of acute cerebral ischemia. The old males gained and then lost weight progressively with age, their pituitary and adrenal glands were heavier than young rats, and their thymi were greatly involuted. The old rats manifested fatty infiltration of the liver and islet beta cell degranulation concomitant with hyperlipidemia, hyperglycemia, and elevated circulating corticosterone. Spontaneous arteriosclerosis was found in these old rats. The acute induction of cerebral ischemia was associated with myocardial infarction in the old but not the young rats.     

Differential effects of aging on the heart rate and blood pressure influences of arterial baroreceptors in awake rats.

The effect of aging on arterial baroreceptor control of heart rate and blood pressure was evaluated in unanesthetized normotensive rats aged 5-6 (young), 12-16 (adult) and 75-90 (old) weeks. Each rat was chronically implanted with arterial and venous femoral catheters and with bilateral balloon-in-cuff occluders around the common carotid arteries. Baroreceptor control of heart rate was assessed by the bradycardic and tachycardic response to intravenous boluses of phenylephrine and nitroprusside, respectively. Carotid baroreceptor control of blood pressure was assessed by a 12-s bilateral common carotid occlusion (CCO). All baroreflex responses were similar in young and adult rats. Compared with the young group, old rats showed a marked reduction of the bradycardic and tachycardic baroreflex response (-42% and -46%, respectively, P less than 0.05). The initial pressor responses to CCO were also impaired in the old animals (3 s: -63%, 6 s: -54%; both P less than 0.01), whereas the peak pressor response (9 and 12 s) was virtually identical in the young and old groups. The preservation of the peak pressor response to CCO in old rats was independent of chemoreceptor activation, aortic baroreceptors or cerebral ischemia. Thus, aging impairs baroreceptor control of heart rate but alters baroreceptor control of blood pressure, as assessed by the pressor response to CCO, only in its fast-developing component, leaving its longer-term component unaffected.