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Effects of Castration on the Senescence of Mice’s Aorta and Intervention Effects of Physiological Testosterone

Author: ZhangLi
Tutor: WuSaiZhu
School: Southern Medical University,
Course: Internal Medicine
Keywords: Castrate Aortic Senescence Testosterone Rb protein Mitochondrial DNA
CLC: R363
Type: Master's thesis
Year: 2011
Downloads: 21
Quote: 0
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Abstract


Aging is a phenomenon of the decline of the general term for the form, structure and function of the entire collective life process, is the inexorable law of life processes, irresistible. When a society 65 years of age or older more than 7% of the total population, in accordance with the standards of the United Nations divided called old age. Fifth census statistics show that in 2000, Chinese elderly people over the age of 65 of 88,834,400, accounting the population structure accounting for 6.8%, consistent with the world average, the 2005 national sample survey of 1% of the population aged 65 and over population of 10,045 people, accounting for 7.69% of the total population, to 2009, the the aging ratio in China has reached 8.3 percent, to catch up with the world average of 7.5%. This shows that China has entered the aging society, and another notable feature of China's population change is \With economic development, improve the quality of life of the population, anti-aging become the focus of attention. Aging-related diseases, cardiovascular disease of the elderly is a not to be neglected. Aging has been the field of life sciences basic and one of the most mysterious research With the rapid development of aging research and molecular biology, the study of the mechanisms of aging from the cellular level, the sub-cellular level gradually toward the molecular and genetic level, is now studies have shown that aging is a process of gene regulation is pre-set, sure as klotho gene anti-aging gene, but aging also influenced by environmental factors and lifestyle. According to statistics, more recognized include aging mechanisms the doctrine of biofilm damage, telomere shortening doctrine, mitochondrial DNA damage doctrine metabolites crosslinked said, somatic mutations that, the error accumulation say, immune disorders, such as free radical damage says so on the free radical theory, mitochondrial DNA damage doctrine, telomere doctrine, the doctrine of biofilm damage genetic program doctrine, chromosome mutation theory, error doctrine, immunity doctrine, endocrine theory. And in 2000 by Franceschi was first proposed inflammatory aging. These doctrines in general can be divided into two categories: one category to ROS, glycosylation, hormone disorders, such as damage to the representatives of environmental damage theory of aging, the other is telomere shortening, cell cycle regulation factors etc. for the program on behalf of the genetic factors theory of aging, the former believe that aging is the result of environmental factors and cumulative destruction of cell; latter that aging is orderly activity of genes in the body, through genetic pre-arranged in accordance with the procedure or the \Various doctrines in-depth discussion of the mechanisms of aging, and embarked on the integration of the theory era, upcoming research focused on environmental factors damage caused by the aging of the genetic program of change within the relevant range. Increase with age, the aging of some of the cells of the vessel wall, endothelial cell morphology and function of the change will occur, the specific cell hypertrophy flat, reduce the secretion of endogenous nitric oxide, showing a proinflammatory and promote bolt of the phenotype, its The structure will be significant changes, including increased vascular stiffness, thickening of the vessel wall, the expansion of the vessel lumen, and decreased vasodilation, these changes have altered the pathophysiological mechanisms of various cardiovascular and cerebrovascular diseases, this may be a variety of aging related diseases, including one of the common mechanism of the formation of aortic atherosclerosis (atherosclerosis, AS). Testosterone is a steroid hormone, androgen male body, is a synthetic hormone, is also a prohormone. Studies have shown that, grow older, the 50-year-old male total testosterone in the body and bioactive testosterone levels gradually decreased. The number of domestic and international studies have shown that sex hormone imbalance is one of the important factors that cause aging, because testosterone to improve the cognitive abilities of the elderly, to adjust the proportion of body fat muscle, increased muscle strength, improved exercise capacity, reduce insulin resistance, inhibit blood lipid abnormalities the increase, improve sexual function, testosterone whether to delay aging deserves further study. Studies have shown that older men plasma testosterone levels are associated with a variety of age-related disease incidence within a certain range, with the decline in testosterone levels, increase the incidence of age-related diseases rise. Studies have shown that a certain concentration of testosterone by the weakening of VCAM-1 and other ways to anti-atherosclerotic role of androgen also has the ability to improve cognitive, inflammatory response, the role of the anti-apoptotic effects, antioxidant system. Us on a topic of research shows that testosterone interfere with the vascular endothelial cells in vitro to inhibit endothelial cell senescence induced by hydrogen peroxide, the system proved that sex hormone imbalance participation and induce the occurrence of aging and development, appropriate to adjust the sex hormone balance can help in anti-aging. But testosterone the possibility of improving or delay aortic aging process, and requires further study. Clear testosterone in the intervention of the aorta in the aging process, expectations provide a new target for age-related prevention and treatment of diseases of the vascular system. In this study, the currently accepted aging markers: (1) in the mouse aorta tissue superoxide dismutase (Superoxide dismutase, SOD) and malondialdehyde (Maleic Dialdehyde, MDA) content. (2) mouse aortic mitochondrial DNA (mtDNA) deletion mutations of the mutation rate. Mitochondrial energy metabolism to proceed from the level of the DNA molecule to investigate the anti-aging method has important significance. (3) its organizational dephosphorylation of Rb protein amount is designed to explore the physiological doses of exogenous testosterone on the mouse aorta aging process in castration. : L Experimental animals and groups of 32 male C57BL/6J mice, 24 8-week-old, 16 were randomly selected castration, another 8 to 18 months of age natural aging C57BL/6J male mice. The grouping follows: normal group (n = 8), ovariectomized group (n = 8), castration physiological dose of testosterone propionate (T) group (n = 8), and the natural aging group (n = 8). 2 production in ovariectomized mice model: removal of the testicles and scrotum stitched randomly selected 16 8-week-old of C57 BL/6J mice. 3 serum testosterone concentration detection: ELISA kit serum testosterone levels in male mice. 4 MDA and SOD concentration detection: MDA thiobarbituric acid, SOD using Oyanagui nitrite formation method. Strictly in accordance with the kit instructions. 5 mice in each group aortic mitochondrial DNA deletion mutations of the mutation rate of detection: extract mitochondrial DNA, in strict accordance with Shenergy gaming genomic DNA extraction kit manual extraction of genomic DNA. Nested PCR and conventional PCR primer design and synthesis, nested PCR and conventional PCR amplification of the corresponding aorta mtDNA fragment. Agarose gel electrophoresis of PCR products and gel analysis system scan results and analysis of the mutation rate. The purified PCR product and to send identification. 6 Western-blot assay phosphorylation of Rb protein expression: total proteins extracted mouse aortic tissue, BCA measurement of protein concentration. 7 Statistical analysis All data were presented as mean ± standard deviation (x ± s), the application spss13.0 standard statistical software, among the groups using single-factor analysis of variance (One-Way ANOVA), linear correlation analysis using line sexual trend test. p lt; 0.05 was considered statistically significant. Results: 1, the mice serum testosterone levels compared with the normal group, the the castrated group with the natural aging mice serum testosterone water on average significantly lower (P = 0.000), and the normal group with castration physiological amounts of testosterone group , between the the castrated group with the natural aging group without statistical differences injection of exogenous testosterone, the castrated mice serum testosterone water average recovery to normal levels. 2, the mice aortic tissue MDA content, SOD activity in comparison with normal mice compared aging group, ovariectomized group aortic tissue MDA content, SOD activity decreased (P = 0.000); given by physiological dose of testosterone after 3 months, MDA content, SOD activity differences had no statistical significance (P gt; 0.05). Aging group, the castration T mice aortic tissue MDA content, SOD activity increased (P = 0.000); castration group aortic MDA content and SOD activity was no significant difference (P = 0.575 and 0.226). PCR agarose gel electrophoresis of four groups of C57 BL/6J mice aortic mitochondrial DNA are amplified 4549bp the unmutated of bands, 916bp, 851bp deletion fragments were also visible. The natural aging group and castration group foreseeable 476bp fragment and 304bp, the castration T group is still visible 304 bp, Compared with normal mice aortic mtDNA mutation rate, castration group with the mutation rate of the natural aging group increased significantly (P = 0.000), and the normal the group with castration T group, the natural aging of the differences between the group with castration group were not statistically significant, the P values ??were 0.220,0.221. That exogenous testosterone propionate, ovariectomized mice aortic mtDNA mutation rate was significantly lower (P = 0.000). Testosterone concentrations mouse aortic mtDNA deletion mutation rate correlation coefficient of 0.720. Results of PCR products were sequenced and used in the second round of the nested PCR primers F3, F4 used to detect fragments of deleted mtDNA sequence, the process is detected by Shanghai Invitrogen Biological Engineering Co., Ltd. (Guangzhou Branch), prove that aging and The ovariectomized mice presence of the the 3713,3864,4236,4415 deletion mutation, their common feature is that both before and after each similar the two sequences. 4, mice aorta to phosphorylation of Rb protein expression in comparison with normal mice compared the castrated mice myocardial tissue dephosphorylation of Rb protein expression was increased (P = 0.001); physiological dose of testosterone intervention months after aortic tissue dephosphorylation of Rb protein expression than the decrease in castrated group (P = 0.001), with no significant difference (P = 0.873) compared to the normal group; natural aging mice myocardial tissue dephosphorylation of Rb protein expression was increased (P = 0.000). Compared with the natural aging mice, castrated mice aorta dephosphorylation of Rb protein expression was not statistically significant (P = 0.410), the castrated group T to phosphorylation of Rb protein expression decreased (P = 0.000 ). Conclusion: 1, C57BL/6J male mice after castration aortic tissue aging process accelerated, three months after its castration aortic degree of aging and natural aging (18 months old) male rats no significant difference; with normal feeding compared to non-castrated male mice, there is a statistically significant difference in the degree of aortic aging. That androgen deficiency accelerates the aging process of mice aorta. 2, and given a physiological dose of exogenous testosterone in male mice, the degree of aortic aging ovariectomized mice a statistically significant difference compared with the normal group, no statistically significant differences in the degree of aging. Exogenous physiological dose of testosterone to slow the mouse aorta aging process.

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