模拟微重力效应对斑马鱼早期胚胎骨质发育的影响

发布时间：2018-02-15 05:58

本文关键词： 模拟微重力效应骨发育细胞应激斑马鱼胚胎　出处：《哈尔滨工业大学》2017年硕士论文　论文类型：学位论文

【摘要】：微重力环境即指在重力或外力作用下引起系统的加速度在10-5~10-4ɡ的环境。目前微重力环境是宇航员在空间飞行和作业中所受到的不可避免的环境刺激。而微重力环境能够引起诸如骨丢失,心脏萎缩,肌肉退化和心脑血管等疾病,严重威胁宇航员的健康。其中微重力环境引起的骨丢失尤为严重,丢失量大,丢失速度快且难以地面恢复,因此研究微重力导致骨丢失的机制十分重要。为了阐明微重力效应对斑马鱼早期胚胎骨质发育的影响,本课题以模式生物斑马鱼为实验材料,微重力效应为研究对象,通过回转技术模拟微重力效应。从形态上观察模拟微重力效应对斑马鱼胚胎发育的影响;利用半定量PCR、Real-Time PCR和全胚原位杂交技术从基因水平上解析模拟微重力效应影响斑马鱼骨质发育的机制。形态学观察与统计分析结果表明,模拟微重力效应可引起斑马鱼早期胚胎死亡率升高、孵化率降低、48 hpf(hour post-fertilization,hpf)心率升高、24 hpf自主运动频率升高,并且还可导致斑马鱼早期胚胎96 hpf发生畸形,畸形类型包括心包水肿,脊柱弯曲和骨折等。茜素红染色结果发现模拟微重力效应可使斑马鱼早期胚胎颅面骨骼:匙骨,副蝶骨,内翼软骨和腮盖骨体积减小,骨量减少,位置改变,说明微重力效应可影响斑马鱼早期胚胎的骨发育和成熟。基因检测结果发现模拟微重力效应可明显抑制斑马鱼早期胚胎骨发育相关基因runx2b和bmp2b的表达,同时氧化应激基因gstp1表达上调。模拟微重力效应还可使活性氧(ROS)和丙二醛(MDA)含量显著上调,并使超氧化物歧化酶(SOD)和过氧化氢酶(CAT)两种抗氧化酶活力上升。说明模拟微重力效应可引起斑马鱼早期胚胎发生氧化应激。同时发现模拟微重力效应还可引起斑马鱼早期胚胎发生内质网应激,可使内质网应激相关基因bip和chop基因表达量显著上调,同时perk基因表达量显著下调,最终导致runx2b基因的上游hmox1a基因表达下调。以上结果表明,模拟微重力效应可通过诱导斑马鱼早期胚胎发生氧化应激和内质网应激抑制hmox1a基因表达,进而抑制runx2b基因的表达,最终影响斑马鱼早期胚胎骨质发育。通过本课题的研究,将明确模拟微重力效应对脊椎动物骨质发育的影响并初步揭示其机制,为宇航员在太空中的健康提供保障,为航空医学领域的研究提供参考。
[Abstract]:Microgravity environment refers to the environment in which the acceleration of the system is 10-5 or 10-4 due to the action of gravity or external force. At present, microgravity environment is an inevitable environmental stimulus for astronauts in space flight and operation. Can cause bone loss, Such diseases as heart atrophy, muscle degeneration and cardiovascular and cerebrovascular diseases seriously threaten the health of astronauts. Bone loss caused by microgravity environment is especially serious, the loss is large, the loss rate is fast and it is difficult to recover on the ground. Therefore, it is very important to study the mechanism of bone loss caused by microgravity. In order to elucidate the effect of microgravity on the bone development of zebrafish early embryo, the model organism zebrafish was used as experimental material, and microgravity effect was used as research object. The effects of simulated microgravity on the embryonic development of zebrafish were observed from the morphological point of view. The mechanism of simulated microgravity effect on bone development of zebrafish was analyzed at gene level by semi-quantitative PCR Real-Time PCR and full-embryo in situ hybridization. The simulated microgravity effect could cause the death rate of zebrafish early embryos to increase, the hatching rate decreased and the heart rate increased by 48 hpf(hour post-fertilization hpf. the frequency of spontaneous motion increased by 24 hpf, and the early zebrafish embryos were deformed at 96 hpf, including pericardial edema. The results of alizarin red staining showed that the simulated microgravity effect could reduce the volume, decrease the bone mass and change the position of the craniofacial bones of zebrafish early embryo, such as spassum bone, accessory sphenoid bone, inner pterygoid cartilage and cheek lid bone. The results showed that the microgravity effect could affect the bone development and maturation of zebrafish early embryos. The results of gene detection showed that simulated microgravity effect could significantly inhibit the expression of bone development related genes runx2b and bmp2b in zebrafish early embryos. At the same time, the expression of oxidative stress gene gstp1 was up-regulated. The simulated microgravity effect also increased the contents of reactive oxygen species (Ros) and malondialdehyde (MDA) significantly. The results showed that the simulated microgravity effect could induce oxidative stress in zebrafish early embryos, and it was also found that simulated microgravity effect could cause spot. Endoplasmic reticulum stress occurs in the early embryos of the horseshoe. The expression of bip and chop genes was up-regulated and the expression of perk gene was down-regulated, which resulted in the down-regulation of hmox1a gene expression in upstream of runx2b gene. The simulated microgravity effect can inhibit the expression of hmox1a gene by inducing oxidative stress and endoplasmic reticulum stress in zebrafish early embryos, and then inhibit the expression of runx2b gene, and ultimately affect the bone development of zebrafish early embryos. The effects of simulated microgravity on vertebrate bone development and its mechanism will be clarified, which will provide a guarantee for the health of astronauts in space and a reference for the research in the field of aeronautical medicine.
【学位授予单位】：哈尔滨工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R856.5

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