【作者】 顾宪敏；

【导师】 马保华；

【作者基本信息】 山东大学 ， 人体解剖与组织胚胎学， 2008， 硕士

【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。Ca²⁺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Ca²⁺浓度的变化主要是通过Ca²⁺转运系统包括存在于质膜及胞内钙库中的钙通道（Calciumchannel）、Ca²⁺泵(Ca²⁺pump)及Ca²⁺/H⁺反向转运子(Ca²⁺/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Ca²⁺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Ca²⁺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Ca²⁺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。更多还原

【Abstract】 Ovarian follicle plays an important role in normal growth and maturity of ovocyte in ovaries. Therefore, Follicular Development Mechanism was specially concerned by reproductive biologists. Ca²⁺ is the very important second messenger in cell signal transduction process, It can regulate a variety of important intracellular enzyme and protein by the calmodulin-binding protein and calcium, There by regulating cell division, differentiation, apoptosis, and so the life course, The change of cytoplasmic free Ca²⁺ concentration achieves mainly through the Ca²⁺ transport system, including the existence of the plasma membrane and intracellular calcium in the calcium channel and Ca²⁺/H⁺ reverse transporter, Among them, There is a calcium channel in the cell membrane or membrane protein macromolecules, the open or close of calcium channel regulate cytoplasmic Ca²⁺concentration,and thus affect certain physiological function of the body cell.This research is about the impact of sodium nitrite particles through the cell membrane calcium channel protein expression can lead to oocytes with the granulosa cells to maintain normal communication of information and material exchange, sodium nitrite, to observe the reproductive toxicity.This paper to study granulosa cell membrane calcium channel protein explain the change of ovarian morphology and express of calcium channel protein ,reveal the reproductive toxicity reaction of sodium nitrite.1、Experiment in vivo: 160 nulliparity female hamsters were randomly divided into experimental group and control group. The experimental group was given sodium nitrite with different concentrations by intraperitoneal injection respectively [2, 4, 8 mg/（kg.d）],and the same volume of normal saline was given to the control group. The changes of granulose cell morphology were observed by light microscopy and electron microscopy.2、Experiment in vitro: the expression of calciphorin was determined by immunoblotting semi-quantitative analysis.3、Mating test: 80 experimental animal mating by 1:1 ratio were observed the mating and reproductive capacity.Results:Experiment in vivo:1. Five days after given sodium nitrite of different concentrations 4mg/（kg.d） and 8mg/（kg.d）, the irregular ovarian follicle and dilatation of blood vessel could be seen in the ovaries of hamster;2. Five days after given sodium nitrite of 8mg/（kg.d） concentration, the pellucida of ovarian follicle became narrow, the microvillus and lipid droplets and the smooth endoplasmic reticulum of granulose cell were reduced definitely;Experiment in vitro: 24 hours after given different concentrations of sodium nitrite, the expressions of the calcium channel proteins of the cultured ovarian granulose cells decreased in a dose-dependent manner.（P<0.05）;Mating test: Five days after given various dosage sodium injection, the birth rate was reduced in a dose-dependent manner.Experimental results show that Choice of the dose of sodium nitrite is a key link in this experiment. When the experimentⅠ,Ⅱ,Ⅲgroup in mice injected with different concentrations of sodium nitrite, Ovarian cells have different changed. When the body load greater concentration of sodium nitrite may affect cell matrix protein synthesis and lead to cell adhesion, migration, changes in location. Therefore, it caused ovarian telangiectasia, congestive particles and the follicle cells are targeted disorder. Sodium nitrite affect the cell membrane Ca²⁺ channel protein expression, And produced a dose-dependent inhibition, Western Blot quantitative analysis test showed that ExperimentalⅠ,Ⅱ,Ⅲprotein were significantly lower in the gradient, experimental groupⅢwas extremely weak positive, and thus affected the Ca²⁺ cells as a cellular signal from the transmission of outward intracellular and interfered with the regulation of physiological activities, With reduced secretion of a transparent, slippery surface cells within the endoplasmic reticulum, mitochondria, fat drops and reduce the cell surface microvilli, Granulosa cells to change shape these oocytes affected the development and maturity, Through the combination of experimental and control fertility experiments show that the mating cage, Sodium nitrite concentration and the ability to control fertility inhibition was directly proportional. Larger dose of sodium nitrite can interfere with the ability to control fertility and have reproductive toxicity.In conclusion, sodium nitrite can significantly effect the normal structure of the ovarian and interfere fertility.更多还原