本文關(guān)鍵詞：關(guān)于亞硝酸鈉對(duì)地鼠卵巢生殖毒性形態(tài)學(xué)相關(guān)性研究　出處：《山東大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 亞硝酸鈉 鈣通道蛋白 卵巢顆粒細(xì)胞 生育能力 地鼠

【摘要】： 卵泡是卵巢內(nèi)卵母細(xì)胞發(fā)育和成熟的微環(huán)境。因此卵泡發(fā)育機(jī)理倍受國(guó)內(nèi)外生殖生物學(xué)家的關(guān)注。Ca~(2+)是細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)過(guò)程中十分重要的第二信使,它直接或間接地通過(guò)鈣調(diào)蛋白及其鈣結(jié)合蛋白調(diào)控細(xì)胞中多種重要的酶及功能蛋白,從而調(diào)節(jié)細(xì)胞分裂、分化、凋亡等生命過(guò)程。胞質(zhì)自由Ca~(2+)濃度的變化主要是通過(guò)Ca~(2+)轉(zhuǎn)運(yùn)系統(tǒng)包括存在于質(zhì)膜及胞內(nèi)鈣庫(kù)中的鈣通道(Calciumchannel)、Ca~(2+)泵(Ca~(2+)pump)及Ca~(2+)/H~+反向轉(zhuǎn)運(yùn)子(Ca~(2+)/H~+antiporter)等的作用來(lái)實(shí)現(xiàn)的。其中,鈣通道是存在于質(zhì)膜或細(xì)胞器膜的大分子蛋白質(zhì)。鈣通道的開放或關(guān)閉調(diào)節(jié)著胞質(zhì)自由Ca~(2+)濃度,從而影響機(jī)體細(xì)胞的某些生理功能。 本實(shí)驗(yàn)是通過(guò)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(xué)和顆粒細(xì)胞質(zhì)膜鈣通道蛋白表達(dá)變化,導(dǎo)致卵母細(xì)胞不能與顆粒細(xì)胞保持正常的信息溝通和物質(zhì)交換,致使卵泡正常發(fā)育成熟和排卵受阻,以此來(lái)觀察亞硝酸鈉的生殖毒性反應(yīng)。本文以孕鼠卵巢形態(tài)學(xué)和粒細(xì)胞質(zhì)膜鈣通道蛋白為研究對(duì)象,闡述了應(yīng)用外源亞硝酸鈉后孕鼠卵巢形態(tài)學(xué)和顆粒細(xì)胞質(zhì)膜鈣通道蛋白表達(dá)水平改變,揭示了亞硝酸鈉在地鼠的生殖毒性作用機(jī)理。 1、體內(nèi)實(shí)驗(yàn):選擇未經(jīng)產(chǎn)雌性成熟金黃地鼠160只,隨機(jī)分為對(duì)照組和實(shí)驗(yàn)組,實(shí)驗(yàn)組分別腹腔注射不同濃度的亞硝酸鈉(2、4、8mg/kg.d),對(duì)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(xì)胞的形態(tài)改變; 2、體外實(shí)驗(yàn):細(xì)胞培養(yǎng)并對(duì)培養(yǎng)的卵巢顆粒細(xì)胞進(jìn)行鈣通道蛋白免疫印跡半定量實(shí)驗(yàn)以觀察鈣通道蛋白的表達(dá)量。 3、合籠交配試驗(yàn)實(shí)驗(yàn)動(dòng)物80只均按1:1比例雌雄合籠行交配試驗(yàn),觀察3個(gè)實(shí)驗(yàn)組動(dòng)物發(fā)情交配和生育能力,對(duì)照組同步做對(duì)照觀察。 體內(nèi)實(shí)驗(yàn)結(jié)果: 1.亞硝酸鈉處理后卵泡的光鏡形態(tài)學(xué)變化給予亞硝酸鈉4mg/(kg.d)和8mg/(kg.d)的負(fù)荷5天后,實(shí)驗(yàn)組Ⅰ剖腹觀察卵泡形態(tài)結(jié)構(gòu)與對(duì)照組動(dòng)物比較變化不大,沒(méi)有統(tǒng)計(jì)意義。但實(shí)驗(yàn)Ⅱ、Ⅲ組停藥后于第6天取卵巢觀察,可見(jiàn)卵巢毛細(xì)血管明顯擴(kuò)張充血,卵泡顆粒細(xì)胞排列紊亂;而對(duì)照組停藥同步觀察,卵巢的形態(tài)結(jié)構(gòu)正常。 2)亞硝酸鈉處理后卵泡的電鏡形態(tài)學(xué)變化給予亞硝酸鈉8mg/(kg.d)負(fù)荷5天后,實(shí)驗(yàn)組Ⅰ組和Ⅱ組取卵巢觀察與對(duì)照組比較沒(méi)有統(tǒng)計(jì)意義。但在實(shí)驗(yàn)Ⅲ組取卵巢觀察,可見(jiàn)透明帶變窄以及顆粒細(xì)胞在透明帶內(nèi)的微絨毛減少,對(duì)照組卵巢觀察可見(jiàn)透明帶以及顆粒細(xì)胞在透明帶中的微絨毛正常;實(shí)驗(yàn)Ⅲ組顆粒細(xì)胞的線粒體、脂滴和滑面內(nèi)質(zhì)網(wǎng)減少;對(duì)照組顆粒細(xì)胞的線粒體、脂滴含量和滑面內(nèi)質(zhì)網(wǎng)表現(xiàn)正常。 體外實(shí)驗(yàn)結(jié)果: 在體外細(xì)胞培養(yǎng)系統(tǒng)內(nèi)追加不同負(fù)荷劑量的亞硝酸鈉24小時(shí)后,培養(yǎng)的顆粒細(xì)胞相應(yīng)的鈣通道蛋白表達(dá)呈計(jì)量依賴性下降(P＜0.05,vs對(duì)照組)。根據(jù)免疫印跡法測(cè)定卵巢顆粒細(xì)胞鈣通道蛋白半定量實(shí)驗(yàn)結(jié)果,卵巢顆粒細(xì)胞鈣通道蛋白表達(dá)(OD,(?)±s):對(duì)照組為81.76±4.33:實(shí)驗(yàn)Ⅰ組為66.49±6.05;實(shí)驗(yàn)Ⅱ組44.42±4.05;實(shí)驗(yàn)組Ⅲ為25.27±3.02,顆粒細(xì)胞鈣通道蛋白含量呈梯度下降。 合籠交配試驗(yàn)結(jié)果: 腹腔內(nèi)注射不同負(fù)荷劑量的亞硝酸鈉5天后,相應(yīng)的地鼠1:1合籠交配后生仔率也呈現(xiàn)計(jì)量依賴性下降。實(shí)驗(yàn)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(duì)照組合籠交配20只,生仔20只;實(shí)驗(yàn)組Ⅰ合籠交配20只,生仔16只;實(shí)驗(yàn)組Ⅱ合籠交配2只,生仔8只;實(shí)驗(yàn)組Ⅲ合籠交配20只,生仔1只,(P＜0.05,vs對(duì)照組)。 上述實(shí)驗(yàn)結(jié)果分析,選擇亞硝酸鈉的劑量濃度是實(shí)驗(yàn)關(guān)鍵環(huán)節(jié)。當(dāng)實(shí)驗(yàn)Ⅰ、Ⅱ、Ⅲ組地鼠接受了不同濃度的亞硝酸鈉時(shí),地鼠卵巢細(xì)胞變化反應(yīng)出現(xiàn)了不同結(jié)果。本實(shí)驗(yàn)認(rèn)為,當(dāng)機(jī)體負(fù)荷較大濃度亞硝酸鈉時(shí)可能會(huì)影響細(xì)胞基質(zhì)蛋白合成而導(dǎo)致細(xì)胞粘著、遷移、定位的改變,因此造成了卵巢毛細(xì)血管擴(kuò)張、充血和卵泡顆粒細(xì)胞排列定位紊亂;亞硝酸鈉影響了細(xì)胞膜Ca~(2+)通道蛋白的表達(dá),出現(xiàn)了濃度依賴性抑制,Western Blot定量分析了實(shí)驗(yàn)Ⅰ、Ⅱ、Ⅲ組蛋白含量,明顯發(fā)生梯度降低;實(shí)驗(yàn)Ⅲ組呈極弱陽(yáng)性表達(dá)(p＜0.01,vs對(duì)照組),從而影響了Ca~(2+)作為細(xì)胞信號(hào)由胞外向胞內(nèi)的傳遞,干擾了細(xì)胞生理調(diào)控活動(dòng),抑制了顆粒細(xì)胞分泌卵膜(zone pellucida)和合成結(jié)構(gòu)蛋白,造成透明帶分泌減少而變窄,細(xì)胞內(nèi)滑面內(nèi)質(zhì)網(wǎng)、線粒體、脂滴和細(xì)胞表面微絨毛減少,顆粒細(xì)胞這些形態(tài)改變影響了卵母細(xì)胞的發(fā)育和成熟。本實(shí)驗(yàn)通過(guò)實(shí)驗(yàn)組和對(duì)照組合籠交配生育實(shí)驗(yàn)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(duì)照組),有生殖毒性作用。 總之,本文主要通過(guò)一系列對(duì)卵巢形態(tài)學(xué)和顆粒細(xì)胞鈣通道蛋白水平上的研究,進(jìn)一步揭示了亞硝酸鈉生殖毒刑的作用機(jī)理,為將來(lái)預(yù)防環(huán)境亞硝酸鈉的影響提供重要的參考和理論實(shí)驗(yàn)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(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R321;R363

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