本文關(guān)鍵詞： 線粒體 線粒體DNA 人 衰老 W1-38細(xì)胞系 PCR γ射線 膜電位 氧化還原酶　出處：《第二軍醫(yī)大學(xué)》2005年碩士論文　論文類型：學(xué)位論文

【摘要】：線粒體作為真核細(xì)胞中一種重要而獨(dú)特的細(xì)胞器,其性狀改變與人的衰老和疾病密切相關(guān),甚至被稱為衰老的生物鐘。自1989年Linnane等提出線粒體衰老假說以來,人們越來越關(guān)注線粒體與衰老的關(guān)系。 因線粒體一些特有的特點(diǎn),如生命周期有限,具有高的更新率;形態(tài)、大小、數(shù)量和分布,常因細(xì)胞種類不同而異;線粒體DNA(mtDNA)與核DNA不同,是多拷貝的且具有異質(zhì)性等,到目前為止,雖對(duì)衰老中線粒體的改變有一些研究報(bào)道,但結(jié)果并不一致,缺乏較系統(tǒng)的研究。關(guān)于人衰老中線粒體的一些變化并無定論,線粒體衰退與機(jī)體的衰老二者的因果關(guān)系也有待于進(jìn)一步探討。機(jī)體的衰老來源于細(xì)胞的衰老,對(duì)二倍體細(xì)胞的研究有助于衰老機(jī)理的探討。通過對(duì)二倍體細(xì)胞衰老中線粒體變化的研究,有助于探討線粒體與衰老的關(guān)系,進(jìn)一步弄清線粒體在衰老中的作用。這些研究將對(duì)衰老指標(biāo)的測定,抗衰老藥物研究及措施的評(píng)估有重要意義。 本實(shí)驗(yàn)主要通過建立細(xì)胞衰老模型,從二倍體細(xì)胞衰老過程中量與功能變化相結(jié)合來探討線粒體與衰老的關(guān)系。 一.人胚肺二倍體細(xì)胞W1-38的培養(yǎng) 近年來應(yīng)用人胚肺二倍體細(xì)胞W1-38作為衰老研究的體外模型,己得到了國內(nèi)外研究工作者的肯定。細(xì)胞從20代傳至約50代;定期凍存;隔期拍照記錄生長狀態(tài);計(jì)數(shù)板計(jì)數(shù);MTT測定細(xì)胞活力。 觀察發(fā)現(xiàn),年輕細(xì)胞平均兩天呈致密單層,細(xì)胞長梭形,排列成束狀,折光度好,立體感強(qiáng);每瓶細(xì)胞數(shù)為6～8×10~5;衰老細(xì)胞形成單層時(shí)間明顯延長,細(xì)胞圓縮蛻變,可呈菱形、三角形,折光度下降;每瓶細(xì)胞數(shù)為4～6×10~5。晚期細(xì)胞活力明顯低于早期。 二.衰老中線粒體質(zhì)和量改變的研究 線粒體形態(tài)因外界環(huán)境和功能改變而異,線粒體形態(tài)變化可作為衰老研究的參考之一。收集固定細(xì)胞,制成切片,電鏡觀察線粒體三維結(jié)構(gòu)和內(nèi)部結(jié)構(gòu)。線粒體的數(shù)量在不同細(xì)胞中差異很大,同時(shí)與細(xì)胞的功能狀態(tài)是相關(guān)的。
[Abstract]:Mitochondria, as an important and unique organelle in eukaryotic cells, are closely related to human aging and disease. Since Linnane et al put forward the hypothesis of mitochondrial senescence in 1989, people have paid more and more attention to the relationship between mitochondria and aging. Because of some unique characteristics of mitochondria, such as limited life cycle, has a high rate of renewal; The shape, size, quantity and distribution of the cells often vary from cell to cell. Mitochondrial DNA (mtDNA), unlike nuclear DNA, is multicopy and heterogeneous. Up to now, there have been some studies on mitochondrial changes in aging, but the results are not consistent. Lack of systematic research. There is no conclusion about some changes of mitochondria in human aging. The causal relationship between mitochondrial decline and aging of organism needs to be further discussed. The senescence of organism comes from cell senescence. The study of diploid cells is helpful to probe into the mechanism of senescence and the relationship between mitochondria and senescence by studying the changes of mitochondria in diploid cells. These studies will be of great significance to the determination of aging indexes, the study of anti-aging drugs and the evaluation of measures. In this experiment, the relationship between mitochondria and senescence was studied by establishing the model of cell senescence and combining the changes of quantity and function during the aging of diploid cells. 1. Culture of human embryonic lung diploid cells W1-38 In recent years, the application of human embryonic lung diploid cell W1-38 as an in vitro model of aging research has been confirmed by researchers at home and abroad. Regular freezing; The growth state was recorded by taking pictures at intervals. Counting board; Cell viability was measured by MTT. The results showed that the young cells were compact monolayer on average for two days with long fusiform cells arranged in bundles good diopter and strong stereosensitivity. The number of cells per bottle was 6 ~ 8 脳 10 ~ (5); The monolayer formation time of senescent cells was obviously prolonged, the cells were round and degenerated, and could be rhomboid, triangular, and diopter decreased. The number of cells per bottle was 4 ~ 6 脳 10 ~ (5). The activity of late stage cells was significantly lower than that of early stage. II. Studies on changes of mitochondrial quality and quantity in aging The morphology of mitochondria varies with the changes of environment and function. The changes of mitochondrial morphology can be used as a reference for the study of aging. Fixed cells are collected and made into sections. Electron microscope was used to observe the three-dimensional and internal structure of mitochondria. The number of mitochondria varied greatly in different cells and was related to the functional state of the cells.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R33

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本文編號(hào)：1451210