| As an important and special organelle in eukaryotic cell, the changes of mitochondrial characters have great relations with human aging and diseases. Sometimes mitochondrion is called aging biological clock. Since mitochondria aging hypothesis was brought forward in 1989 by Linnane, people pay more attention to the correlation between mitochondria and aging.Mitochondria have some special characters, such as limited life cycle; high turnover rate; different shapes, sizes, quantity and distributing. And mitochondrial DNA(mtDNA) is different from nuclear DNA, many copies and having heterplasmy. Because of these characters, up to now, there are some reports about mitochondrial changes during aging process, but the results are not consistent, lacking systemic studies. There is still no the last word about mitochondrial changes in human senescence, and the consequence between mitochoindrial decaying and organism aging is waited to research further. Organism aging comes from cell aging, so study on diploid cell line is helpful to research the mechanism of aging. By the study of mitochondrial changes during aging, we can search after the relationship between mitochondria and aging, and make more clear about the role of mitochondria during aging. These studies will do much to the detection of aging signs, the manufacture of the drugs and evaluation of measures to resist aging.Our study focused on research the correlation between mitochondria and aging by combining the quantity and function changes of diploid cell line with age via cell model.1. cell cultureThese years, using human diploid cell line as research aging model in vitro has been accepted by scholars in world. In our study, the WI-38 human diploid cell line was derived from normal embryonic (3 months gestation) lung tissue. The cells were cultured from 20 to 50 passage number, punctual cryopreservation, and recorded growing state by taking photos at intervals. The cells were taken count using a haemocytometer, and the viability was determined by MTT.We found the young cells were about 2 days to form monolayer, long shuttle-like, bunchily arranged, bright and better third dimension. It was 6~8X105 cells after forming monolayer . The aging cells forming monolayer prolonged, shrinking, taking on rhombus and triangle, declining refraction. It was 4~6X105 cells after forming monolayer. And the viability declined obviously with age.2. Study on changes of mitochondrial quantity and function during aging processMitochondrial shapes change with the environment and function, so the shapes changes may be looked as one of the parameters to aging. We collected and fixuped the cells, made slices, then observed the mitochondrial spatial configuration using electron microscope. Mitochondrial quantity is different from each kind of cells, and it is correlative to the action. We isolated mitochondria by differential velocity centrifugation, the content of mitochondrial protein was determined using BCA-100 Protein Quantitative Analysis Kit. Different from nuclear DNA, mtDNA has many copies and has a close relationship with function. So it is significative to study the content changes of mtDNA with age. Using 18srDNA of nuclear as an internal standard, therelative amount of mtDNA was determined by a competitive polymerase chain reaction(PCR) method. Marked with Digoxigenin(Dig), PCR formed probes, Point blotting analysed the content of mtDNA, to validate the results of PCR.. The energy metabolizability decline is a remarkable character of organism aging. As the power factory of cell, mitochondrion provides the most energy for life action. The function changes have great relationship with aging. Labelled with [3H]TdR, we determined the updating rate of mtDNA. Dyed with R123 and TMRM, mitochondrial membrane potential was measured by flow cytometry(FLl channels). Isolated by differential velocity centrifugation, mitochondrial oxidoreductase activity was measured by Absorbance Microplate Reader.Having the young cell compared with the senile, we found mitochondrial membrane potential and oxidoreductase activity of the senile cells declined, but mitochondrial quantity and mtDNA content were increased. Analyzing the possible mechanisms, we think there mainly has two. One is a feedback mechanism , namely, the age-related increase of mitochondrial content may be a feedback mechanism to compensate for the function decline of mitochondria. The other is a quickened replication mechanism, namely, short and mutational mtDNA has the advantage to reproduce, making the deficient mitochondria increased.3. Study on changes of mitochondrial quantity and function in diploid human cell line WI-38 irradiated by 60Co y —rayThe former studies on diseases caused by radiation mostly observed the damnification of nuclear DNA, less attention to the mtDNA. In prophase research, we have found that the D-loop region of mtDNA.appeard obvious break points after cells irradiated by 60Co y — ray(20Gy). If...
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