發(fā)布時間：2018-01-13 06:15

  本文關鍵詞：關于亞硝酸鈉對地鼠卵巢生殖毒性形態(tài)學相關性研究　出處：《山東大學》2008年碩士論文　論文類型：學位論文

  更多相關文章： 亞硝酸鈉 鈣通道蛋白 卵巢顆粒細胞 生育能力 地鼠

【摘要】： 卵泡是卵巢內卵母細胞發(fā)育和成熟的微環(huán)境。因此卵泡發(fā)育機理倍受國內外生殖生物學家的關注。Ca~(2+)是細胞信號轉導過程中十分重要的第二信使,它直接或間接地通過鈣調蛋白及其鈣結合蛋白調控細胞中多種重要的酶及功能蛋白,從而調節(jié)細胞分裂、分化、凋亡等生命過程。胞質自由Ca~(2+)濃度的變化主要是通過Ca~(2+)轉運系統(tǒng)包括存在于質膜及胞內鈣庫中的鈣通道(Calciumchannel)、Ca~(2+)泵(Ca~(2+)pump)及Ca~(2+)/H~+反向轉運子(Ca~(2+)/H~+antiporter)等的作用來實現(xiàn)的。其中,鈣通道是存在于質膜或細胞器膜的大分子蛋白質。鈣通道的開放或關閉調節(jié)著胞質自由Ca~(2+)濃度,從而影響機體細胞的某些生理功能。 本實驗是通過外界環(huán)境因素亞硝酸鈉(Sodium nitrite)的作用,影響孕鼠卵巢形態(tài)學和顆粒細胞質膜鈣通道蛋白表達變化,導致卵母細胞不能與顆粒細胞保持正常的信息溝通和物質交換,致使卵泡正常發(fā)育成熟和排卵受阻,以此來觀察亞硝酸鈉的生殖毒性反應。本文以孕鼠卵巢形態(tài)學和粒細胞質膜鈣通道蛋白為研究對象,闡述了應用外源亞硝酸鈉后孕鼠卵巢形態(tài)學和顆粒細胞質膜鈣通道蛋白表達水平改變,揭示了亞硝酸鈉在地鼠的生殖毒性作用機理。 1、體內實驗:選擇未經(jīng)產(chǎn)雌性成熟金黃地鼠160只,隨機分為對照組和實驗組,實驗組分別腹腔注射不同濃度的亞硝酸鈉(2、4、8mg/kg.d),對照組給予生理鹽水。采用光鏡及電鏡的方法觀察卵泡及顆粒細胞的形態(tài)改變; 2、體外實驗:細胞培養(yǎng)并對培養(yǎng)的卵巢顆粒細胞進行鈣通道蛋白免疫印跡半定量實驗以觀察鈣通道蛋白的表達量。 3、合籠交配試驗實驗動物80只均按1:1比例雌雄合籠行交配試驗,觀察3個實驗組動物發(fā)情交配和生育能力,對照組同步做對照觀察。 體內實驗結果: 1.亞硝酸鈉處理后卵泡的光鏡形態(tài)學變化給予亞硝酸鈉4mg/(kg.d)和8mg/(kg.d)的負荷5天后,實驗組Ⅰ剖腹觀察卵泡形態(tài)結構與對照組動物比較變化不大,沒有統(tǒng)計意義。但實驗Ⅱ、Ⅲ組停藥后于第6天取卵巢觀察,可見卵巢毛細血管明顯擴張充血,卵泡顆粒細胞排列紊亂;而對照組停藥同步觀察,卵巢的形態(tài)結構正常。 2)亞硝酸鈉處理后卵泡的電鏡形態(tài)學變化給予亞硝酸鈉8mg/(kg.d)負荷5天后,實驗組Ⅰ組和Ⅱ組取卵巢觀察與對照組比較沒有統(tǒng)計意義。但在實驗Ⅲ組取卵巢觀察,可見透明帶變窄以及顆粒細胞在透明帶內的微絨毛減少,對照組卵巢觀察可見透明帶以及顆粒細胞在透明帶中的微絨毛正常;實驗Ⅲ組顆粒細胞的線粒體、脂滴和滑面內質網(wǎng)減少;對照組顆粒細胞的線粒體、脂滴含量和滑面內質網(wǎng)表現(xiàn)正常。 體外實驗結果: 在體外細胞培養(yǎng)系統(tǒng)內追加不同負荷劑量的亞硝酸鈉24小時后,培養(yǎng)的顆粒細胞相應的鈣通道蛋白表達呈計量依賴性下降(P＜0.05,vs對照組)。根據(jù)免疫印跡法測定卵巢顆粒細胞鈣通道蛋白半定量實驗結果,卵巢顆粒細胞鈣通道蛋白表達(OD,(?)±s):對照組為81.76±4.33:實驗Ⅰ組為66.49±6.05;實驗Ⅱ組44.42±4.05;實驗組Ⅲ為25.27±3.02,顆粒細胞鈣通道蛋白含量呈梯度下降。 合籠交配試驗結果: 腹腔內注射不同負荷劑量的亞硝酸鈉5天后,相應的地鼠1:1合籠交配后生仔率也呈現(xiàn)計量依賴性下降。實驗組和生理鹽水組之間的生育情況有明顯的差異(P＞0.05)。注射亞硝酸鈉后生育抑制情況如下:對照組合籠交配20只,生仔20只;實驗組Ⅰ合籠交配20只,生仔16只;實驗組Ⅱ合籠交配2只,生仔8只;實驗組Ⅲ合籠交配20只,生仔1只,(P＜0.05,vs對照組)。 上述實驗結果分析,選擇亞硝酸鈉的劑量濃度是實驗關鍵環(huán)節(jié)。當實驗Ⅰ、Ⅱ、Ⅲ組地鼠接受了不同濃度的亞硝酸鈉時,地鼠卵巢細胞變化反應出現(xiàn)了不同結果。本實驗認為,當機體負荷較大濃度亞硝酸鈉時可能會影響細胞基質蛋白合成而導致細胞粘著、遷移、定位的改變,因此造成了卵巢毛細血管擴張、充血和卵泡顆粒細胞排列定位紊亂;亞硝酸鈉影響了細胞膜Ca~(2+)通道蛋白的表達,出現(xiàn)了濃度依賴性抑制,Western Blot定量分析了實驗Ⅰ、Ⅱ、Ⅲ組蛋白含量,明顯發(fā)生梯度降低;實驗Ⅲ組呈極弱陽性表達(p＜0.01,vs對照組),從而影響了Ca~(2+)作為細胞信號由胞外向胞內的傳遞,干擾了細胞生理調控活動,抑制了顆粒細胞分泌卵膜(zone pellucida)和合成結構蛋白,造成透明帶分泌減少而變窄,細胞內滑面內質網(wǎng)、線粒體、脂滴和細胞表面微絨毛減少,顆粒細胞這些形態(tài)改變影響了卵母細胞的發(fā)育和成熟。本實驗通過實驗組和對照組合籠交配生育實驗表明,亞硝酸鈉濃度與地鼠生育能力抑制呈正比。較大劑量的亞硝酸鈉能干擾地鼠生育能力(p＜0.05,vs對照組),有生殖毒性作用。 總之,本文主要通過一系列對卵巢形態(tài)學和顆粒細胞鈣通道蛋白水平上的研究,進一步揭示了亞硝酸鈉生殖毒刑的作用機理,為將來預防環(huán)境亞硝酸鈉的影響提供重要的參考和理論實驗依據(jù)。
[Abstract]:Is the development of follicular oocytes and mature microenvironment. Therefore follicular development mechanism specially concerned by reproductive biologists.Ca~ (2+) is a very important signal transduction in second messenger, which directly or indirectly through the calcium binding and calmodulin enzyme and protein function of several important protein regulatory cells. In order to regulate cell division, differentiation, apoptosis and other life processes. The cytosolic free Ca~ (2+) concentration is mainly through Ca~ (2+) transport system including calcium channels are present in the plasma membrane and intracellular calcium in the library (Calciumchannel), Ca~ (2+) pump (Ca~ (2+) pump and Ca~ (2+) reverse transporter (Ca~) /H~+ (2+) /H~+antiporter), to achieve the effect. The calcium channel protein in the plasma membrane or organelle membrane. Calcium channel regulates the opening or closing of the cytosolic free Ca~ (2+) concentration, thus affecting the human body Some physiological functions.
This experiment of sodium nitrite environmental factors (Sodium nitrite) the role of the influence of calcium channel in pregnant rats ovarian morphology and cytoplasm membrane protein expression changes, resulting in oocytes and granulosa cells can maintain normal communication of information and material exchange, resulting in normal follicular maturation and ovulation was blocked, in order to observe the reproductive toxicity sodium nitrite. The calcium channel of pregnant rats ovarian morphology and grain cytoplasmic membrane protein as the research object, elaborated the calcium channel of pregnant rats ovarian morphology and cytoplasm membrane protein expression changes after application of exogenous sodium nitrite, sodium nitrite revealed in the reproductive toxicity mechanism of hamsters.
1, the in vivo experiment: nulliparous female mature hamster 160 rats were randomly divided into control group and experimental group, different concentrations of sodium nitrite in experimental group were intraperitoneal injection (2,4,8mg/kg.d), the control group received saline. By light microscopy and electron microscopy observation method and particle morphology of the follicle cell change;
2, in vitro experiment: cell culture and the semi quantitative test of calcium channel protein immunoblotting for cultured ovarian granulosa cells to observe the expression of calcium channel protein.
3, 80 mating animals were tested according to 1:1 ratio. The mating and fertility of 3 groups were observed.
The results of the experiment in vivo:
Given the sodium nitrite 4mg/ light microscopic morphological changes of follicular 1. sodium nitrite after treatment (kg.d) and 8mg/ (kg.d) load after 5 days, the experimental group of exploratory laparotomy was performed to observe the morphological structure and the follicle control group animal relatively little change, no statistical significance. But the experiment group II, III after discontinuation of the ovaries was observed in sixth day, the ovarian capillaries dilated, granulosa cells arranged in disorder; while the control group stopped synchronous observation, the morphology and structure of ovary was normal.
2) given sodium nitrite 8mg/ morphology change of follicles after treatment of sodium nitrite (kg.d) load after 5 days, the experimental group of group I and group II ovaries observed compared with the control group was not statistically significant. But in the experimental group with the ovaries observed, hyaline with narrowed and granular cell reduction in transparent zone the control group was observed on ovarian granulosa cells in the zona pellucida and the microvilli of normal granulosa cells; experimental group of mitochondria and smooth endoplasmic reticulum, lipid droplets decreased; the control group of granulosa cell mitochondria, lipid content and smooth endoplasmic reticulum showed normal.
In vitro experiment results:
In vitro cell culture system of sodium nitrite given different loading dose after 24 hours, the expression of calcium channel protein in granulosa cells cultured in a dose-dependent manner. The corresponding decreased (P < 0.05 vs, control group). According to the experimental results of semi quantitative determination of ovarian granulosa cell calcium channel protein by Western blot, the expression of ovarian granulosa cells calcium channel protein (OD, (?) + s): the control group was 81.76 + 4.33: in experiment group is 66.49 + 6.05; 44.42 + 4.05 experimental group II; experimental group III was 25.27 + 3.02, granular calcium channel protein content showed a gradient descent.
Caged mating test results:
Intraperitoneal injection of sodium nitrite in different loading dose 5 days later, the hamster 1:1 mating epigenetic rate also showed a measurement dependent decline. Fertility between the experimental group and the saline group had significant difference (P > 0.05). Sodium nitrite injection after fertility inhibition as follows: control combined cage mating 20, birth 20; experimental group I mated 20, yes 16; the experimental group II mated with 2, 8 children; the experimental group III mated 20, foaled 1, (P = 0.05, vs group).
Analysis of the experimental results, choose the dose of sodium nitrite is a key link in this experiment. When the experiment I, II, III sodium nitrite group injected with different concentrations, reaction changes of hamster ovary cell appeared different results. According to this experiment, when may affect cell matrix protein synthesis and lead to cell adhesion and migration body load greater concentration of sodium nitrite, positioning changes, thus causing ovarian telangiectasia, congestion and granulosa cells arranged positioning disorder; sodium nitrite affected the cell membrane Ca~ (2+) channel protein expression showed a concentration dependent inhibition, Western Blot quantitative analysis experiment I, II, III group of protein content, significantly reduce gradient; experimental group showed very weak positive expression (P < 0.01, vs control group), thus affecting the Ca~ (2+) as cell signal from cell outward intracellular delivery, interfere with cell physiology Control activities, inhibition of granulosa cells in the egg membrane (zone pellucida) and the synthesis of structural proteins, resulting in reduced secretion of zona pellucida and narrow cells of smooth endoplasmic reticulum, mitochondria, lipid droplets and cell surface microvilli, change the granulosa cells of these morphological influences the development and maturation of oocyte. Through this experiment, the experimental group and the the control group were mated fertility experiments showed that sodium nitrite concentration and the inhibition was proportional to the hamster fertility. Larger dose of sodium nitrite can interfere fertility (P < 0.05, vs control group), reproductive toxicity.
In conclusion, this paper mainly through a series of studies on ovarian granulosa cell morphology and calcium channel protein level, further reveals the mechanism of sodium nitrite reproductive torture, provide an important reference and theoretical basis for the future prevention effect of sodium nitrite in the environment.

【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R321;R363

【參考文獻】

相關期刊論文 前10條

1 李俊杰,賈青,田樹軍;雌性哺乳動物LH及其受體的研究進展[J];黃牛雜志;2003年03期

2 林雪玲,盧玉葵;小鼠卵巢顆粒細胞條件培養(yǎng)液中蛋白質和激素水平[J];佛山科學技術學院學報(自然科學版);2000年03期

3 湯熙翔,夏家輝;鈣通道與人類遺傳病[J];國外醫(yī)學.遺傳學分冊;2000年03期

4 李智文,張樂,王麗娜;硝酸鹽、亞硝酸鹽及N-亞硝基化合物與人類先天畸形[J];環(huán)境與健康雜志;2005年06期

5 陳曉宇,劉東,李青旺,張德福,李震,于瑞嵩,劉惠莉,周宗清;豬卵巢卵母細胞的收集和體外成熟培養(yǎng)[J];上海農業(yè)學報;2003年02期

6 鄭斐,呂時銘;白介素-6調節(jié)顆粒細胞雌孕激素分泌的作用[J];生殖與避孕;2001年02期

7 霍立軍,楊增明;哺乳動物黃體退化的分子調控[J];生殖醫(yī)學雜志;2002年03期

8 李智文;任愛國;關聯(lián)欣;李竹;;山西省出生缺陷高發(fā)區(qū)居民飲水中硝酸鹽和亞硝酸鹽含量研究[J];衛(wèi)生研究;2006年02期

9 石玉華;陳子江;張鵬;盛燕;高芹;;不孕患者卵巢功能早衰病因分析[J];現(xiàn)代婦產(chǎn)科進展;2007年03期

10 顧憲敏;馬保華;禹卉;董平;;亞硝酸鈉對地鼠生殖毒性的研究[J];現(xiàn)代婦產(chǎn)科進展;2008年04期

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