心脏端粒酶的研究概况

端粒长度、端粒酶活性在心血管疾病发生发展过程中的变化引起了心血管界的关注。现就端粒酶在正常心脏中的表达及活性的变化,端粒酶与心肌细胞损伤、冠状动脉粥样硬化、心力衰竭等心血管疾病之间的关系和检测心脏端粒长度和端粒酶活性的技术和方法作一综述。     

Telomerase and the aging process

The level of telomerase activity is important in determining telomere length in aging cells and tissues. Here evidence on the importance of telomerase activity is reviewed with respect to aging rates of mammalian species and the health and life span of individuals within a species. The significance of telomerase reactivation for both cancer development and for immortalizing cells for therapeutic processes is assessed.     

Decline in oxygen consumption correlates with lifespan in long-lived and short-lived mutants of Caenorhabditis elegans

In humans, the basal energy metabolism is thought to decline linearly with age. On the other hand, in the nematode Caenorhabditis elegans, two research groups reported independently that it declined exponentially. In this study, furthermore, we used various lifespan-mutant strains to determine whether the previous conclusion is more likely to be true. We can indirectly estimate the metabolic energy by conveniently measuring the oxygen consumption rates of C. elegans using an optical apparatus. From the profile of respiratory rates as a function of age, we can quantitatively isolate the physiological decline rate, λ, that exponentially represents the decay rate of respiratory activity with age. In addition, quantitative analysis indicates that the respiratory activity of worms has a finite value in advanced age. We also show that the maximum and mean lifespans strongly correlate with the reciprocal of the λ. These findings offer crucial biochemical evidence for a molecular mechanism at work in biological aging. Consequently, we here propose a mechanism based on a chemical reaction and offer a definition of the physiological decline rate and the finiteness of respiratory activity in advanced age.     

Validated analysis of mortality rates demonstrates distinct genetic mechanisms that influence lifespan

A key goal of gerontology is to discover the factors that influence the rate of senescence, which in this context refers to the age-dependent acceleration of mortality, inversely related to the morality rate doubling time. In contrast factors that influence only initial mortality rate are thought to be less relevant to the fundamental processes of aging. To resolve these two determinants of mortality rate and lifespan, initial morality rate and rate of senescence are calculated using the Gompertz equation. Despite theoretical and empirical evidence that the Gompertz parameters are most consistently and reliably estimated by maximum-likelihood techniques, and somewhat less so by non-linear regression, many researchers continue to use linear regression on the log-transformed hazard rate. The present study compares these three methods in the analysis of several published studies. Estimates of the mortality rate parameters were then used to compare the theoretical values to the actual values of the following parameters: maximal lifespan, 50% survival times, variance in control groups and agreement with the distribution of deaths. These comparisons indicate that maximum-likelihood and non-linear regression estimates provide better estimates of mortality rate parameters than log-linear regression. Of particular interest, the improved estimates indicate that most genetic manipulations in mice that increase lifespan do so by decreasing initial mortality rate, not rate of senescence, whereas most genetic manipulations that decrease lifespan surprisingly do so by increasing the rate of senescence, not initial mortality rate.     

A critical period in lifespan of male rats coincides with increased oxidative stress

The oxidative stress theory of aging has provided the best possible explanation for the processes which accompany aging and has received much support, however, in the last few years there have been questions regarding the validity of this theory. We have conducted experiments to determine an array of oxidative stress parameters in blood of male rats at various intervals (1, 4, 8, 12, 18 and 24 months) during their entire lifespan. Established protocols were used to measure plasma antioxidant capacity, erythrocyte plasma membrane redox system (PMRS), lipid and protein oxidation in erythrocytes and plasma, and erythrocyte glutathione (GSH). Our results on the total plasma antioxidant potential, PMRS in erythrocytes, protein and lipid peroxidation, and intracellular reduced GSH provide evidence that oxidative stress is minimal till approximately one-third of the total lifespan (8 months) and there is a spurt in oxidative stress between 8 and 12 months. The identification of a period (corresponding to 8–12 months) in the lifespan of rats coinciding with an spurt in oxidative stress is an interesting finding. No such report is available in humans or in any other model systems during aging.     

淫羊藿苷延长秀丽线虫寿命与机制

目的探讨淫羊藿苷（Icariin,Ica）对秀丽线虫寿命的干预效果与机制,为筛选延缓衰老、延长寿命的药物提供实验依据。方法将秀丽线虫分为空白对照组、Ica5mg／L、10mg／L、15mg／L、20mg／L各剂量干预实验组,观察在Ica不同梯度剂量作用下秀丽线虫寿命的变化,以明确Ica作用的最佳剂量。接着设立秀丽线虫空白对照组和Ica（15mg／L）干预组,评价Ica对秀丽线虫平均寿命、生殖能力的干预效果;同时进行急性热应激和急性氧化应激实验,观察应激时线虫的平均生存时间,探讨Ica延长线虫寿命的机制。结果15mg／L的Ica是延长秀丽线虫寿命的最佳剂量。Ica组线虫的平均寿命明显长于空白对照组[（31．87±4．46）d vs．（20．13±2．81）d]（P〈0．01）;Ica组线虫生殖高峰期子代数目比空白对照组显著增加（151±21．13VS．122±17．09,P〈O．01）;急性热应激实验和急性氧化应激实验发现Ica组线虫平均生存时间均明显长于空白对照组[（9．53±1．34）hVS．（5．97±0．84）h,（8．34±1．17）hvs.（5．77±O．81）h]（P〈0．01）。结论淫羊藿苷能延长秀丽线虫的寿命,其机制可能与淫羊藿苷提高线虫的应激能力有关。     

In vivo analysis of gene expression in long-lived mice lacking the pregnancy-associated plasma protein A (PappA) gene

Mice lacking the pregnancy-associated plasma protein A (PappA) gene exhibit diminished localized IGF-1 bioavailability and a 30% increase in mean life span. However, it is uncertain which tissues exhibit reduced IGF-1 signals in the PappA(−/−) mouse, and whether effects of this mutation parallel those of mutations that diminish IGF-1 in serum. Across a panel of 21 tissues, we used RT-PCR to evaluate the effects of the PappA(−/−) mutation on expression of Igfbp5, which served as an in vivo indicator of IGF-1 signaling. Among these tissues, expression of Igfbp5 was significantly reduced by PappA(−/−) only in kidney. A broader survey of IGF-associated genes in six organs identified five other genes responsive to PappA(−/−) in kidney, with stronger effects in this organ relative to other tissues. Renal expression of Irs1 and Mt1 was increased by PappA(−/−) as well as by mutations that reduce IGF-1 in serum (i.e., Ghr(−/−), Pit1(dw/dw) and Prop1(df/df)), and we demonstrate that expression of these genes is regulated by growth hormone-treatment and calorie restriction. These results provide in vivo data on an important new model of mammalian aging, and characterize both similar and contrasting expression patterns between long-lived mice with reduced local IGF-1 availability and diminished IGF-1 in serum.     

Clinical usefulness of telomerase activity and telomere length in the preoperative diagnosis of gastric and colorectal cancer

It has been reported that telomerase activity and telomeric reduction can be detected in many human cancers. Although it is well known that telomerase activity and telomere length have important implications for cancer biology, their clinical usefulness in the preoperative diagnosis of gastric and colorectal cancer has not been elucidated. Therefore, we examined telomerase activity and telomere length in gastric and colorectal cancer using tissue samples obtained by fiberscopy. Telomerase activity was measured by a telomeric repeat amplification protocol (TRAP). Although telomerase activity was detected in 1/12 (8%) cases of gastric polyp and in 2/9 (22%) cases of colorectal polyp, its positivity in gastric cancer and colorectal cancer was 7/10 (70%) and 21/26 (81%; P < 0.0003 and P < 0.0001, respectively). Telomere length was analyzed by Southern blotting, and telomeric reduction in gastric cancer was significantly greater than that in gastric polyp (P < 0.0003). However, there was no telomeric reduction between colorectal cancer and colorectal polyp. The results of the present study indicate that determination of telomerase activity and telomere length may serve as a useful method for preoperative diagnosis of gastric and colorectal cancer. Received: 16 October 1998 / Accepted: 2 February 1999     

膀胱癌组织端粒酶活性表达及其与临床病理特征关系的研究

目的 探讨膀胱移行细胞癌（transitional cells carcinoma,TCC）组织端粒酶活性表达与膀胱癌临床病理特征的关系.方法 应用端粒酶重复序列扩增（telomeric repeat amplification protocol,TRAP）银染TRAP-ELISA方法检测25例膀胱癌组织（实验组）和4例膀胱正常组织（对照组）中端粒酶活性的表达,并与临床病理分期、病理分级及预后关系进行分析.结果 实验组中端粒酶活性表达率为96%,对照组无表达,实验组端粒酶活性高于对照组（P＜0.001）;端粒酶活性临床病理分期T2高于T1、T3高于T2（P＜0.01）,晚期组高于早期组（P＜0.001）;端粒酶活性病理分级G2高于G1、G3高于G2（P＜0.01）.结论 实验组端粒酶活性高表达,并与膀胱癌临床病理分期、病理分级呈正相关,端粒酶激活可能在膀胱癌的发生及发展过程中起重要作用,端粒酶可作为膀胱癌早期诊断及判定预后的分子生物学标记.     

Telomeric 3'-overhang length is associated with the size of telomeres

Telomeres are specialized DNA/protein complexes that cap eukaryotic chromosome ends as T-loop structures and maintain genomic integrity. Vertebrate telomeric DNA consists of tandem double-strand repeats which terminate in a 3' single-strand G-rich overhang. The telomeric 3'-overhang is important for the formation of the T-loop. In mammalian mortal somatic cells, telomeres shorten with each successive division and contribute to the onset of replicative senescence. The exact molecular mechanism underlying replicative senescence remains unclear: whether telomere shortening is the only trigger or loss of telomeric 3'-overhang plays a causal role. To further address this issue, we investigated telomeric 3'-overhang and telomere changes during cell proliferation toward replicative senescence. We demonstrate here that telomeric 3'-overhang, similar to telomeres, exhibits progressive attrition with each cell division in primary sheep fibroblasts and that telomeric 3'-overhang size does not determine the rate of telomere shortening. Furthermore, the sizes of telomeric 3'-overhangs are associated with telomere lengths. Our results suggest that alteration of the 3'-overhang and the telomere during cellular proliferation are associated. Together they may contribute to maintain chromosomal stability and to regulate replicative senescence.     

免疫功能受损宿主外周血单个核细胞端粒酶活性研究

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Telomerase activity in colorectal cancer, prognostic factor and implications in the microsatellite instability pathway

AIM： To determine whether the telomerase activity is related to the Microsatellite instability （MSI） genetic pathway and whether it means a difference in the survival.
METHODS： The population consisted of 97 colorectal cancer patients. MSI determination was performed in accordance with the NCI criteria using PCR and Genescan. Telomerase activity was determined by the TRAP-assay, an ELISA procedure based on the amplification of telomeric repeat sequences.
RESULTS： 6.2% showed high MSI （MSI-H）, 10.3% showed low MSI （MSI-L） and 83.5% did not show this alteration （MSS）. Positive telomerase activity was detected in 92.8% of the patients. 83.3% of MSI-H tumors showed positive telomerase against 93.8% of MSS tumors. In the overall survival analysis the absence of telomerase activity conferred a better prognosis.
CONCLUSION： Previous works have shown that tumors which develop via the MSI pathway present a better prognosis. No link between telomerase activity and MSl status is observed, although sample sizes are small. Patients with telomerase negative tumors had better overall survival than patients with telomerase positive tumors.     

A further test of the equation of lifespan by C. elegans: effective activation energy for aging and lifespan

We previously proposed a rate theory of chemical reaction as well as a lifespan equation derived by a stochastic fluctuation theory. Both were applied to biodemographic data by C. elegans to quantitatively explain that respiratory activity declines exponentially with age and that it has a physiological decline rate and a finite value (threshold) in advanced age. In this work, using the poikilothermic nature of Caenorhabditis elegans, we demonstrate the further validity of the rate theory of chemical reaction as well as the lifespan equation by changing two methods. First, to test the appropriateness of the lifespan equation from another aspect, lifespan assays were conducted by varying the time interval of observation employing the egl-1 mutant. The results indicate that, as the time interval is reduced, mortality rates gradually approach the force of mortality expected from the fitting equation of the survival curve. Second, based on the dependence of lifespan on the temperature of the culture, the physiological decline rate, and the onset of biodemographic aging, we show that the effective activation energy or energy barrier for aging and lifespan may be closely related to the standard free-energy change of ATP or ADP for a wild type and some lifespan-related mutants of C. elegans.     

The functional VNTR MNS16A of the TERT gene is associated with human longevity in a population of Central Italy

Background

Telomerase, encoded by TERT, is the ribonucleoprotein polymerase that maintains telomere ends and it plays a crucial role in cellular senescence. TERT single nucleotide polymorphisms (SNPs) have been associated both with various malignancies and telomere length (TL). The association of TERT SNPs with longevity remains uncertain and varies with ethnicity. The aim of this study was to investigate whether the functional variable number of tandem repeat (VNTR) MNS16A of TERT is associated with longevity.

Methods

MNS16A genotypes have been determined for 1072 unrelated healthy individuals from Central Italy (18–106 years old) divided into three gender-specific age classes defined according to demographic information and accounting for the different survivals between sexes: for men (women), the first class consists of individuals < 66 years old (< 73 years old), the second class of individuals 66–88 years old (73–91 years old), and the third class of individuals > 88 years old (> 91 years old). TL was assessed using genomic DNA from whole blood of 72 selected individuals by a multiplex real-time PCR assay.

Results

MNS16A appears associated to longevity, showing significant associations in Comparison 2 (Age Class 3 vs. Age Class 2) under both additive (odds ratio [O.R.] 0.749; p = 0.019) and dominant (O.R. 0.579; p = 0.011) models. The MNS16A*L allele is significantly underrepresented in Age Class 3 (O.R. 0.759; p = 0.020) compared to Age Class 2. A significant telomere attrition is reported along the three age classes (p = 0.0001), that remains significant only in L*/L* genotype carriers (p = 0.002) when the analysis was conducted according to MNS16A genotype.

Conclusions

The TERT MNS16A*L allele appears negatively associated with longevity. The concomitant significant telomere cross sectional attrition rate observed for L*/L* genotype suggests that this polymorphism could influence human longevity by affecting TL.     

Extended longevity of queen honey bees compared to workers is associated with peroxidation-resistant membranes

In the honey bee (Apis mellifera), depending on what they are fed, female eggs become either workers or queens. Although queens and workers share a common genome, the maximum lifespan of queens is an order-of-magnitude longer than workers. The mechanistic basis of this longevity difference is unknown. In order to test if differences in membrane composition could be involved we have compared the fatty acid composition of phospholipids of queen and worker honey bees. The cell membranes of both young and old honey bee queens are highly monounsaturated with very low content of polyunsaturates. Newly emerged workers have a similar membrane fatty acid composition to queens but within the first week of hive life, they increase the polyunsaturate content and decrease the monounsaturate content of their membranes, probably as a result of pollen consumption. This means their membranes likely become more susceptible to lipid peroxidation in this first week of hive life. The results support the suggestion that membrane composition might be an important factor in the determination of maximum lifespan. Assuming the same slope of the relationship between membrane peroxidation index and maximum lifespan as previously observed for mammal and bird species, we propose that the 3-fold difference in peroxidation index of phospholipids of queens and workers is large enough to account for the order-of-magnitude difference in their longevity.     

Low resting metabolic rate is associated with greater lifespan because of a confounding effect of body fatness

A negative association between resting metabolic rate (RMR) and lifespan is the cornerstone of the rate of living and free-radical damage theories of aging. Empirical studies supporting a negative association of RMR to lifespan may arise from the correlation between RMR and both daily energy expenditure (DEE) and thermoregulatory activity energy expenditure (TAEE). We screened 540 female mice for higher and lower DEE and measured RMR in the resulting 324 (60 %). We then selected 92 mice in which there was no link between residual from the regression of RMR against body mass (BM) and residual of DEE against BM to separate the effects of these traits. Lifespan was not significantly related to body mass, DEE and TAEE, but significantly negatively related to RMR. Fat-free mass (FFM) and fat mass (FM) were both significantly positively related to RMR. After removing the effect of FFM on RMR, the association between RMR and lifespan remained significantly negative; however, after statistically removing the effect of FM on RMR, the significant association between RMR and lifespan disappeared. We conclude that the negative association between RMR and lifespan is primarily due to the effect of FM, with FM positively related to both RMR and mortality and hence RMR negatively to lifespan. In 40 additional screened mice, greater FM was also associated with greater oxidative damage to DNA.