【作者】 于永生；

【导师】 苏定冯；

【作者基本信息】 第二军医大学 ， 药理学， 2010， 博士

【摘要】 超重(hypergravity)现象一直是航空航天人员面临的主要问题之一。超重其物理定义就是物体对支持物的压力或对悬绳的拉力大于物体所承受重力的现象。宇宙飞船在起飞和再入大气层阶段时航天员都处于超重状态。现有的文献的表明在进行宇宙空间飞行时,机体发生了一系列生理和病理的改变。然而,有限的飞行机会和宇宙空间飞行过程中出现的其他非重力影响因素要求我们建立一个在地面使用人工重力模拟超重的模型来研究超重对机体生理的影响。本课题我们采用了国际比较认可的“离心机模型”来模拟超重现象。有文献报道,宇宙空间飞行对雄性生殖生理存在许多不利影响。相应的“离心机模型”实验中也发现一定程度的超重刺激会持续抑制睾酮分泌和精子的正常生成。然而,其中的发生机制尚为被阐明,随着宇宙空间开发力度的增加,中国载人航空的发展,新的登月计划的开始以及在可预见的将来人类的月球定居,关于超重对人类生殖系统的损伤作用,及其发病机制,寻找用于治疗和预防这种损伤的药物靶点都是亟待解决的问题。本课题主要研究内容包括:1.利用“离心机模型”研究超重刺激对雄性小鼠生殖系统生理功能的危害。本实验表明在一定强度的超重(3.3G)刺激下,小鼠的精子数目和活力都随着刺激次数的增加而减少,5天后变化达到显著程度,而在这个过程中睾丸和体重的变化不明显;在超重刺激5天后,用western blot实验检测凋亡相关蛋白caspase-3,促凋亡因子bax和抑制凋亡因子bcl-2的表达,发现睾丸细胞发生了明显的凋亡,Tunel实验表明凋亡主要发生在睾丸间质细胞和精原细胞;同时检测到血清中睾酮含量明显下降,但小鼠的性行为能力指标“捕捉次数”变化不明显。2.找到超重刺激后小鼠睾丸中所出现的一个重要的差异蛋白“swiprosin-1”(SWP);并验证和明确该蛋白和超重的关系。在超重刺激下,睾丸中SWP蛋白的表达随着刺激时间的延长而增加,并且随着刺激强度的增加而增加;免疫组化实验显示这种蛋白在小鼠睾丸中主要分布在睾丸间质细胞(Leydig cell)。3.明确该蛋白超重刺激后表达变化并非因为应激引起的。考虑到接受超重刺激后的动物其本身也处于应激状态,为了确定SWP在睾丸中的表达变化并不是由于动物处于应激状态下所出现的非特异性改变,我们给予动物各种常见的应激刺激(冷,热和缺氧)后发现,SWP在睾丸中的表达并没有发生明显改变;在细胞水平,应用长效、强效的糖皮质激素类药物“地塞米松”和小鼠特有的“应激激素”皮质酮来作用于原代培养的小鼠睾丸间质细胞,SWP蛋白的表达并没有发生明显改变。这些结果提示我们SWP蛋白在睾丸的表达上调对于超重刺激具有特异性。4.研究该蛋白的表达变化对小鼠生殖系统的影响。通过使用由慢病毒包装的质粒转染小鼠睾丸间质细胞的方法在睾丸局部注射该病毒,并以空白病毒作为阴性对照;当SWP表达被干扰后,局部SWP表达下调,发现精子数目和精子活力明显下降;同样方法,使睾丸局部SWP蛋白的表达上调后,发现精子数目和精子活力也明显下降了。首先告诉我们超重刺激后SWP蛋白的表达上调造成了对生殖系统的损伤,其次也提示了SWP蛋白的作用并非简单地单向地影响小鼠生殖系统的功能,其表达水平在睾丸中有适宜量,超过或低于这个表达量都会对生殖系统造成不利影响。5.研究该蛋白与Leydig细胞凋亡之间的关系。培养原代的Leydig细胞,通过将质粒转染入细胞,干扰和过表达SWP蛋白的表达,通过检测caspase-3剪切体和前体,bax和bcl-2蛋白的表达,结果显示SWP蛋白低表达时睾丸间质细胞的凋亡减少,而SWP蛋白高表达时,凋亡的发生减少。6.研究该蛋白与一氧化氮之间的关系。培养原代的Leydig细胞,通过将质粒转染入细胞,干扰和过表达SWP蛋白的表达,结果显示SWP蛋白低表达时睾丸间质细胞的iNOS表达增加,相应的NO的产生增加而SWP蛋白高表达时,iNOS表达下降,NO的产生减少,而eNOS表达在两种情况下变化都不明显。7.研究该蛋白与睾酮合成之间的关系。培养原代的Leydig细胞,通过质粒转染细胞,干扰和过表达SWP蛋白的表达,结果显示SWP蛋白低表达时睾丸间质细胞分泌的睾酮增加,而SWP蛋白高表达时,睾酮的浓度明显下降,同时检测了细胞内cAMP的含量,未检测到明显变化,提示SWP对睾酮的分泌有影响但并非通过影响cAMP。综上所述,在超重刺激作用下,雄性小鼠睾丸中SWP蛋白的表达增加,通过排除了这是超重刺激时所伴随的应激引起这种蛋白表达发生变化的可能性,认为这种蛋白的表达上调对于超重刺激具有特异性。同时也发现SWP的表达增加可以损伤雄性成年小鼠的生殖系统功能。其中睾丸间质细胞凋亡的增加以及其分泌睾酮的减少可能是SWP蛋白的作用机制,而睾丸中SWP蛋白表达下调时精子受到损伤的主要原因是iNOS的表达增加后睾丸中NO含量的增加所导致。本课题为预防和治疗因为超重引起的生殖系统损伤找到了一个重要靶标蛋白。更多还原

【Abstract】 Background and objectivesA major goal of space life sciences research is to broaden scientific knowledge of the influence of gravity on living systems. Investigators have known for decades that the spaceflight environment can have dramatic effects on reproductive system. However, there are only limited reports on the mechanisms behind these changes.The spaceflight environment includes at least three factors that likely influence reproduction. These include mission-related psychological stress, low-dose-to-low-dose rate radiation, and changes in inertial condition (i.e., microgravity and hypergravity).Mammalian reproduction evolved within Earth’s 1-g gravitational field. Therefore, deviations from Earth’s normal gravity, i.e., hypogravity (forces<1 g) or hypergravity (forces>1 g), may compromise reproduction. Lift-off and re-entry of the spacecraft are associated with exposure to increased gravity (hypergravity). However, limited spaceflight opportunities and confounding effects of various nongravitational factors associated with spaceflight (i.e., radiation, stress) have led to the development of ground-based models for studying the effects of altered gravity on biological systems. Centrifugation is used to study hypergravity.The present study was designed to investigate effects of hypergravity on reproduction of male mouse and the mechanisms behind it.Methods and results1. In the mice subjected to hypergravity (3.3G) in the centrifuge, the number or motility of sperm, testosterone concentration of serum and NO-production of testis was decreased and the apoptosis of testicular cells was increased significantly. But the body weight, testis weight and capture rate did not change significantly.2. A novel protein, swiprosin-1(SWP) was found highly expressed in testis after mice suffered hypergravity and the expression increased with the times and intensity of hypergravity. The SWP was found distributed in the leydig cells in testis with Immunohistochemistry analysis.3. The expression of SWP in mice testis has no significant decreased after three different kinds of stress, hypoxia, hot, and hypothermia. In the primary cultured leydig cells, neither dexamethasone nor corticosterone did change the expression of SWP.4. The number or motility of sperm decreased after swiprosin-1 RNAi and also decreased after SWP overproduction through direct injection of recombinant lentivirus into mouse testis.5. The apoptosis in cultured leydig cells was decreased in after SWP RNAi and increased after SWP over-expression according to the expression of caspase-3, bax and bcl-2.6. The concentration of NO of the supernatant of cultured leydig cells and expression of iNOS increased after SWP RNAi, decreased after SWP overproduction.7. The concentration of testosterone of the supernatant of cultured leddig cells decreased after SWP over-expression, increased after SWP RNAi.ConclusionAugmentation of SWP expression attributes to hypergravity-induced reproductive injury in mouse. The protein-SWP may be an important target for prevention or treatment for hypergravity-induced reproductive injury.更多还原