本文選題：鎘 + 小鼠��； 參考：《揚州大學》2017年碩士論文

【摘要】：近年來,鎘的毒性研究一直受到人們的廣泛關(guān)注。機體攝入過量的鎘可以引起多種器官和組織細胞的損傷,除主要危害肝臟、腎臟和骨骼外,對生殖系統(tǒng)也會造成損傷,例如危害精子的發(fā)生,改變卵巢的形態(tài)結(jié)構(gòu),抑制卵泡的生長與發(fā)育等。但是,環(huán)境鎘暴露對卵母細胞以及后代的危害尚不完全清楚。飲用含鎘量過高的水是機體攝入過量鎘的一種主要方式。本研究以ICR小鼠連續(xù)35天自由飲用含鎘水(64mg/L的CdCl2·2.5H2O)復制動物模型,研究鎘對小鼠卵母細胞成熟以及早期胚胎發(fā)育的影響。1.鎘對小鼠卵母細胞成熟的影響為了消除自來水中鎘的影響,對照組雌鼠飲用Milli-Q超純水,實驗組雌鼠飲用Milli-Q水配置的含鎘水。通過超數(shù)排卵實驗,檢測卵泡的發(fā)育和成熟,體內(nèi)和體外實驗檢測卵母細胞的成熟情況,應用免疫熒光方法檢測卵母細胞成熟過程中染色體、紡錘體、肌動蛋白帽、線粒體的形態(tài)和功能,并檢測卵母細胞內(nèi)ATP含量的變化;通過檢測卵母細胞內(nèi)組蛋白H3K9甲基化和H4K12乙�；絹矸磻k對表觀遺傳修飾的影響。結(jié)果表明,鎘處理導致雌性小鼠超排后卵母細胞數(shù)量的顯著減少(p0.05);體內(nèi)和體外實驗均表明,鎘處理后卵母細胞生發(fā)泡破裂(GVBD)無變化,但是大部分卵母細胞停滯在第一次減數(shù)分裂中期(MⅠ期),與對照組相比,成熟卵母細胞(MⅡ)所占比例顯著下降,說明鎘處理不會抑制卵母細胞減數(shù)分裂的恢復,但干擾了第一次減數(shù)分裂的正常完成。免疫熒光顯示,鎘處理導致MⅡ期卵母細胞染色體、紡錘體和細胞極性的異常,鎘處理后MⅡ期卵母細胞的染色體并未整齊排列,紡錘體形態(tài)沒有呈現(xiàn)典型的梭形,同時Actin cap出現(xiàn)丟失,說明鎘處理導致MⅡ期卵母細胞的質(zhì)量出現(xiàn)下降。進一步檢測了線粒體的數(shù)量和功能,結(jié)果表明,鎘處理導致MⅡ期卵母細胞中線粒體數(shù)目出現(xiàn)顯著下降,雖然線粒體功能得到了增強,但卻未能挽救ATP含量的下降。組蛋白修飾的檢測結(jié)果顯示,鎘處理顯著增強了 MⅡ期卵母細胞H3K9的甲基化水平和H4K12的乙�；�。通過孤雌激活實驗來觀察鎘對卵母細胞第二次減數(shù)分裂的影響,結(jié)果顯示,雖然鎘處理延緩了第二極體的排出和原核的形成,但并未改變最終形成原核的數(shù)目,說明鎘處理并未顯著影響第二次減數(shù)分裂的最終完成。2.鎘對小鼠早期胚胎發(fā)育的影響將鎘處理的雌鼠與正常雄鼠交配后,采集受精卵進行體外培養(yǎng),以觀察鎘對早期胚胎發(fā)育的影響;結(jié)果顯示,鎘處理顯著降低了同一時期4細胞胚胎和囊胚的發(fā)育率。在進一步的實驗中,采集鎘處理雌鼠的卵母細胞進行體外受精,與對照組相比,囊胚發(fā)育率同樣下降。這說明鎘處理降低了成熟卵母細胞的質(zhì)量,并進一步影響了其受精后早期胚胎的正常發(fā)育。綜上所述,飲水型慢性鎘中毒不僅會降低雌性小鼠卵母細胞排卵率,而且通過影響減數(shù)分裂進程、線粒體功能和組蛋白修飾來降低卵母細胞的質(zhì)量,并最終導致胚胎發(fā)育能力的下降。
[Abstract]:In recent years, the toxicity of cadmium has been widely concerned. Excessive intake of cadmium can cause damage to various organs and tissue cells. In addition to the main harm to the liver, kidney and bone, it can also cause damage to the reproductive system, such as the occurrence of sperm, the morphologic changes of the ovaries, the inhibition of the growth and development of follicles. However, the harm of environmental cadmium exposure to oocytes and offspring is not completely clear. Drinking too high cadmium content water is a major way of excessive cadmium intake. In this study, ICR mice were free to drink cadmium water (64mg/L CdCl2 / 2.5H2O) for 35 days to copy animal models, and to study the maturation of oocytes and early embryos of mice by cadmium. The effect of.1. cadmium on the maturation of mouse oocyte in order to eliminate the effect of cadmium in the tap water. The female rats in the control group drank the ultra pure water of Milli-Q, and the female rats in the experimental group drank the cadmium water in the Milli-Q water. The development and maturation of the follicles were detected by the superovulation experiments. The maturation of oocyte in vivo and in vitro should be tested in vivo and in vitro. Detection of chromosomes, spindles, actin caps, morphologies and functions of mitochondria during oocyte maturation and detection of changes in ATP content in oocytes by immunofluorescence, and the effects of cadmium on epigenetic modification by detecting the levels of H3K9 methylation and H4K12 acetylation in oocytes. The number of oocytes in female mice decreased significantly (P0.05), and in vitro and in vitro experiments showed that there was no change in the germinal vesicle rupture (GVBD) of oocytes after cadmium treatment, but most oocytes were stagnated at the middle of the first meiosis (M I), and the proportion of mature oocytes (M II) decreased significantly compared with those in the group. Cadmium treatment does not inhibit the restoration of meiosis in oocyte, but interferes with the normal completion of the first meiosis. Immunofluorescence shows that cadmium treatment leads to the chromosomes of M II oocyte, the abnormal spindle and cell polarity, and the chromosomes of M II oocytes after cadmium treatment are not arranged neatly, and the spindle shape is not typical. At the same time, the loss of Actin cap showed that cadmium treatment resulted in a decrease in the quality of M II oocytes. The number and function of mitochondria were further detected. The results showed that the number of mitochondria in M II oocytes decreased significantly and the mitochondrial function was enhanced, but the content of ATP was not saved. Declines. The results of histone modification showed that cadmium treatment significantly enhanced the methylation level of H3K9 in M II oocyte and the level of acetylation of H4K12. The effects of cadmium on the second meiosis of oocytes were observed by parthenogenetic activation. The results showed that although cadmium treatment postpones the discharge of the second polar body and the formation of the prokaryotic cells, The effect of cadmium treatment did not change the number of prokaryotic cells, indicating that cadmium treatment did not significantly affect the second meiotic division. The effect of cadmium on the development of early embryo in mice was not significantly affected by the effect of cadmium on the development of early embryos in mice. After mating with normal male rats, the female mice were collected by the cadmium treated mouse and cultured in vitro to observe the effect of cadmium on the development of the early embryos. The results showed that cadmium treatment showed that cadmium treatment showed that cadmium treatment was significant. The development rate of 4 cell embryos and blastocysts in the same period was reduced. In further experiments, the oocytes of the female rats were fertilized in vitro. Compared with the control group, the development rate of blastocyst was also decreased. This indicates that cadmium treatment reduces the quality of mature oocytes and affects the normal hair of the early embryos after their fertilization. In summary, drinking water type chronic cadmium poisoning not only reduces the ovulation rate of oocyte in female mice, but also affects the meiosis process, mitochondrial function and histone modification to reduce the quality of oocyte, and eventually lead to the decline of embryonic development ability.
【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S859.8

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