| [Objective]Aging is the general term which includes the total gradual decline process of life form, structure and function,this phenomenon is the inevitable law of life course which is also irresistible.By the United Nations standards, when a society has more than 7% of the total population whose age more than 65 years old, called it the old type. According to statistics, the fifth census in 2000, there are 88,834,400 of our Chinese people over the age of 65, take the part of 6.8% of the whole population, consistent with the world's average,1% of the national population sample survey in 2005 showed that there were 10,045 people over 65 years old taking the ratio of7.69% of our total population, and in the year 2009, the proportion of the aging of China has reached 8.3%, surpass the world average of 7.5 significantly.In 2000 the elderly population aged 65 and above coefficient from 4.91% in 1982 gradually increased to 5.57% in 1990, and even 6.96% in 2000. From those data, we found that China has entered the aging society, and the changes in China's population has a distinctive feature which calls "old before getting rich. With the development of economic and people's life quality, aging becomes more attractive and attracts our society's focus of attention. And so do the age-related diseases,especially the cardiovascular disease which caused by aging can not be ignored.Aging has always been one of the most fundamental and the most mysterious researches in life science.With the rapid development of molecular biology,mechanism of aging research has developped from the cellular level, subcellular level to molecular and genetic level step by step. immunological theory, endocrine theory, and so on. At preset the Studies reveal that aging is a process of gene regulation which is pre-set; currently the anti-aging gene named klotho gene etc supported by many experiments, such as and other genes, but aging is also affected by the impact of environmental factors and lifestyle Acording to statistics,the research of aging has made great progress and a number of excellent theories have occurred, such as free radical theory, membrane damage theory, mitochondrial DNA mutagenesis theory,telomere shortening theory, genetic programming theory, chromosome mutation theory, error theory, theory of the immune, endocrine theory and so on, and the inflammatory aging first proposed by Franceschi et al in 2000.Generally speaking, these theories can be divided into two major categories:one is damage theory, regarding aging as the results of the damage from environment such as ROS, glycosylation, hormonal disorders, and so on; the other is genetic program theory based on telomere shortening, cell cycle regulation factors, considering aging is genetically pre-arranged program characterized by over expression of "aging" genes and low expression of "young" genes. Recently, the two theories become to cooperate gradually, and further study will focus on the combined effects of damage from environmental factors and genetic program, as well as the interaction of them.Testosterone is one of the steroid hormone, the main androgen of a man, which is a synthetic hormone, and a prohormone. Studies have shown that total testosterone and the testosterone which has biological activity will decline gradually and obviously in man's vivo when he is more than 50 years old male body and biological activity of testosterone, total levels decline gradually and significantly. A number of domestic and international research shows that hormone imbalance is one of the important factors causing aging, many exogenous testosterone replacement therapy clinical trial foundlt is also the arduous battle we human have to fight against especially when we are approaching an aging society. Many studies indicate that androgen disequilibrium is one of the key factors triggering aging. Aging can cause many problems, such as Loss of muscle strength, heart attack,and so on, but wether the arduous can delay the process of aging needs futher research.lt has been suggested that the decline of sexual hormones is closely related to the aging process,. And, properly regulating gonadal hormone equilibrium can delay the aging process. Recent researches proved testosterone can carry out anti-AS effect by attenuating VCAM-1 and through many other approaches. Androgen can improve cognitive capacity, lessen inflammatory reaction, improve anti-languor effect and anti-oxidation effect.As time pass by, some cell of blood vessel wall becomes decrepit, and the form and fuction of vessel wall change gravely,including cells become flat and mast, the decline of endogenous nitric oxide, and showing a pro-inflammatory phenotype and promote the bolt.The structure also changes a lot, including luminal enlargement, initimal. and medial thickening, increased vascular stiffness, partial vascular cells (including vescular endothelial cells) aging, and the endothelium-dependent vasodilator respones which are estimates of endothelial function declines.However, so far, no literature has been concerned about the effect of androgen deletion and extrinsic physiological doses of testosterone on the aging process of mice'aortic vessel wall tissues. The research adopts established aging indicators, namely, the amount of SOD and MDA in mice'aorta; mutation rate of mtDNA deletion in mice'aorta, and the dephosphorylation Rb protein expression. The research is aimed at observing the effect of the physiological doses of testosterone on the aging of aorta in mice of the castrated group. The research adopts established aging indicators, namely, the amount of SOD and MDA in mice'aorta; mutation rate of mtDNA deletion in mice'aorta, and the dephosphorylation Rb protein expression. The research is aimed at observing the effect of the physiological doses of testosterone on the aging of aorta in mice of the castrated group.[Methods]1. laboratory animals Thirty two C57BL/6J male mice, Twenty four of the mice were eight weeks old, sixteenof the mice were randomly separate from the mice, while the remaining eight ones that age naturally were eighteen month old. Animal grouping as follows:the normal groups(n=8), the castrated group (n=8), and the castrated+physiological doses of testosterone(T)(n=8), the naturally aging group(n=8)2. Castrated mice modeling:16 of the Eight-week-old C57 BL/6J mice were randomly choosed, and then testicles were cut and the scrotums were stitched.3.The measure of serum testosterone concentration:ELISA kit was used to measure serum testosterone level.4.Measurement of MDA and SOD concentration:Thiobarbituric acid was utilized to measure MDA. The technique of Oyannagui's nitrite formation was applied to measure SOD. The measurement of SOD AND MDA concentration should be carried out strictly in line with the kit specification.5. Measurement of mutation rate of aortic mt DNA deletion of the three groups DNA extraction should be strictly in line with DNA Abstraction Kit Speculation stipulated. Primers were designed and synthesised. Nested PCR was applied to amplify deletion-type mtDNA fragments and general PCR was applied wild-type mtDNA fragments. The agarose gel electrophoresis identification and the mutation rate analysis of PCR products.Then the optical density integral was recorded at very mutant respectively.After that, The identification and the sequencing of PCR products6.Western-blot was utilized to measure dephosphorylation Rb protein expression, Totle protein was extracted using protein issue extract. Protein concentration was measured using BCA method.7.All data were indicated by mean±standard deviation(x±s. Spssl3.0 was used for the research. One—Way ANOVA was employed for each interclass. Liner trend analysis was adopted for liner correlation analysis. If p is <0.05, the result bears statistical significance.[results]1. Each group's serum testosterone level Compared with the normal group, both the castrated group and the naturally aged group display a significant drop in serum testosterone level. Besides, no statistical difference can be found between the normal group and the castrated +physiology doses of testosterone group, between the castrated group and the naturally aged group. Having been injected with extrinsic testosterone, the castrated group have their serum testosterone level recovered to the normal level2.comparison of MDA amount and SOD activity between each group's aortas Compared with the normal group, the naturally aged group and the castrated group have higher MDA amount but lower SOD activity(P=0.000 in both situations).Three months after being given physiological doses of testosterone, the naturally aged group and the castrated group display no statistical significance in MDA amount and SOD activity((P>0.05).Compared with the naturally aged group, the castrated +T group have lower MDA amount in aorta but higher SOD activity ((P=0.000 in both situations).There is no statistical significance in the difference of aortic MDA amount and SOD activity(P=0.575,P=0.226 respectively)3.Result of PCR by agarose gel electrophoresis Aortic mtDNA in four groups of C57 BL/6J mice all amplify to have 4549bp non-mutation straps and can detect 916bp,851bp deletion fragments.476bp fragments and 304bp can be detected in the castrated group and the naturally aged group, while 304bp can be found in the castrated+T group. Compared with the mutation rate of aortic mtDNA of the normal group, that of both the castrated group and the naturally aged group increase significantly ((P=0.000).Besides, no statistical significance can be found between the castrated+T group and the normal group, between the naturally aged group and the castrated group(p=0.220, p=0.221 respectively).Having been given extrinsic testosterone propionate, the castrated group find its aortic mtDNA mutation rate dropping significantly(p=0.000). The coefficient between testosterone concentration and mice'aortic mtDNA deletion mutation rate is 0.720. The result of PCR product sequencing. Primers F3, F4 used in the second round of Nested PCR were applied to detect the sequence of deletion-type fragments, the process of which was detected by Guangzhou Office, Invitrogen(Shanghai).The detection prove that the naturally aged group and the castrated group have 3713,3864,4236,4415 detection mutation, the common feature of which is that two similar two series of sequences existe prior to or post the deletion. 4. The comparison of the dephosphorylation Rb protein expression in each group's aortas In comparison with the normal group, the dephosphorylation Rb protein expression in the castrated group's cardiac muscle is higher (P=0.001).Three months after being given physiological doses of testosterone, the castrated +T group aortic tissues'dephosphorylation Rb protein expression is lower (P=0.001) in comparison to the castrated group, but bears no statistical differences((P=0.000) with the castrated group. Compared with the naturally aged group, aortic dephosphorylation Rb protein expression in the castrated group bears no statistical significance(P=0.410). The dephosphorylation Rb protein expression in the castrated +T group is lower((P=0.000)[Conclusion]1,Lack of testosterone can speed up the C57BL/6J male mice's aortic aging process, and has no significant difference between the castrated group and natural aging aorta (18 months); Otherwise,compares with normal group, the result is in contrast.That is say, androgen deficiency accelerates the aging process in mice aorta2,When give the castrated mice physiological doses of testosterone, their aorta's aging process were delayed, and the differences between the the castrated group and the castrated+T group were significant. In other words,extrinsic physiological doses of testosterone delays aging process of aortas. |
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