关于亚硝酸钠对地鼠卵巢生殖毒性形态学相关性研究

发布时间：2018-01-13 06:15

本文关键词：关于亚硝酸钠对地鼠卵巢生殖毒性形态学相关性研究　出处：《山东大学》2008年硕士论文　论文类型：学位论文

【摘要】： 卵泡是卵巢内卵母细胞发育和成熟的微环境。因此卵泡发育机理倍受国内外生殖生物学家的关注。Ca~(2+)是细胞信号转导过程中十分重要的第二信使,它直接或间接地通过钙调蛋白及其钙结合蛋白调控细胞中多种重要的酶及功能蛋白,从而调节细胞分裂、分化、凋亡等生命过程。胞质自由Ca~(2+)浓度的变化主要是通过Ca~(2+)转运系统包括存在于质膜及胞内钙库中的钙通道(Calciumchannel)、Ca~(2+)泵(Ca~(2+)pump)及Ca~(2+)/H~+反向转运子(Ca~(2+)/H~+antiporter)等的作用来实现的。其中,钙通道是存在于质膜或细胞器膜的大分子蛋白质。钙通道的开放或关闭调节着胞质自由Ca~(2+)浓度,从而影响机体细胞的某些生理功能。本实验是通过外界环境因素亚硝酸钠(Sodium nitrite)的作用,影响孕鼠卵巢形态学和颗粒细胞质膜钙通道蛋白表达变化,导致卵母细胞不能与颗粒细胞保持正常的信息沟通和物质交换,致使卵泡正常发育成熟和排卵受阻,以此来观察亚硝酸钠的生殖毒性反应。本文以孕鼠卵巢形态学和粒细胞质膜钙通道蛋白为研究对象,阐述了应用外源亚硝酸钠后孕鼠卵巢形态学和颗粒细胞质膜钙通道蛋白表达水平改变,揭示了亚硝酸钠在地鼠的生殖毒性作用机理。 1、体内实验:选择未经产雌性成熟金黄地鼠160只,随机分为对照组和实验组,实验组分别腹腔注射不同浓度的亚硝酸钠(2、4、8mg/kg.d),对照组给予生理盐水。采用光镜及电镜的方法观察卵泡及颗粒细胞的形态改变; 2、体外实验:细胞培养并对培养的卵巢颗粒细胞进行钙通道蛋白免疫印迹半定量实验以观察钙通道蛋白的表达量。 3、合笼交配试验实验动物80只均按1:1比例雌雄合笼行交配试验,观察3个实验组动物发情交配和生育能力,对照组同步做对照观察。体内实验结果: 1.亚硝酸钠处理后卵泡的光镜形态学变化给予亚硝酸钠4mg/(kg.d)和8mg/(kg.d)的负荷5天后,实验组Ⅰ剖腹观察卵泡形态结构与对照组动物比较变化不大,没有统计意义。但实验Ⅱ、Ⅲ组停药后于第6天取卵巢观察,可见卵巢毛细血管明显扩张充血,卵泡颗粒细胞排列紊乱;而对照组停药同步观察,卵巢的形态结构正常。 2)亚硝酸钠处理后卵泡的电镜形态学变化给予亚硝酸钠8mg/(kg.d)负荷5天后,实验组Ⅰ组和Ⅱ组取卵巢观察与对照组比较没有统计意义。但在实验Ⅲ组取卵巢观察,可见透明带变窄以及颗粒细胞在透明带内的微绒毛减少,对照组卵巢观察可见透明带以及颗粒细胞在透明带中的微绒毛正常;实验Ⅲ组颗粒细胞的线粒体、脂滴和滑面内质网减少;对照组颗粒细胞的线粒体、脂滴含量和滑面内质网表现正常。体外实验结果: 在体外细胞培养系统内追加不同负荷剂量的亚硝酸钠24小时后,培养的颗粒细胞相应的钙通道蛋白表达呈计量依赖性下降(P＜0.05,vs对照组)。根据免疫印迹法测定卵巢颗粒细胞钙通道蛋白半定量实验结果,卵巢颗粒细胞钙通道蛋白表达(OD,(?)±s):对照组为81.76±4.33:实验Ⅰ组为66.49±6.05;实验Ⅱ组44.42±4.05;实验组Ⅲ为25.27±3.02,颗粒细胞钙通道蛋白含量呈梯度下降。合笼交配试验结果: 腹腔内注射不同负荷剂量的亚硝酸钠5天后,相应的地鼠1:1合笼交配后生仔率也呈现计量依赖性下降。实验组和生理盐水组之间的生育情况有明显的差异(P＞0.05)。注射亚硝酸钠后生育抑制情况如下:对照组合笼交配20只,生仔20只;实验组Ⅰ合笼交配20只,生仔16只;实验组Ⅱ合笼交配2只,生仔8只;实验组Ⅲ合笼交配20只,生仔1只,(P＜0.05,vs对照组)。上述实验结果分析,选择亚硝酸钠的剂量浓度是实验关键环节。当实验Ⅰ、Ⅱ、Ⅲ组地鼠接受了不同浓度的亚硝酸钠时,地鼠卵巢细胞变化反应出现了不同结果。本实验认为,当机体负荷较大浓度亚硝酸钠时可能会影响细胞基质蛋白合成而导致细胞粘着、迁移、定位的改变,因此造成了卵巢毛细血管扩张、充血和卵泡颗粒细胞排列定位紊乱;亚硝酸钠影响了细胞膜Ca~(2+)通道蛋白的表达,出现了浓度依赖性抑制,Western Blot定量分析了实验Ⅰ、Ⅱ、Ⅲ组蛋白含量,明显发生梯度降低;实验Ⅲ组呈极弱阳性表达(p＜0.01,vs对照组),从而影响了Ca~(2+)作为细胞信号由胞外向胞内的传递,干扰了细胞生理调控活动,抑制了颗粒细胞分泌卵膜(zone pellucida)和合成结构蛋白,造成透明带分泌减少而变窄,细胞内滑面内质网、线粒体、脂滴和细胞表面微绒毛减少,颗粒细胞这些形态改变影响了卵母细胞的发育和成熟。本实验通过实验组和对照组合笼交配生育实验表明,亚硝酸钠浓度与地鼠生育能力抑制呈正比。较大剂量的亚硝酸钠能干扰地鼠生育能力(p＜0.05,vs对照组),有生殖毒性作用。总之,本文主要通过一系列对卵巢形态学和颗粒细胞钙通道蛋白水平上的研究,进一步揭示了亚硝酸钠生殖毒刑的作用机理,为将来预防环境亚硝酸钠的影响提供重要的参考和理论实验依据。
[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.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R321;R363

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