大通牦牛心肌组织低氧适应发育学研究

发布时间：2018-12-30 13:49

【摘要】：为探讨在高原低氧环境下不同发育期大通牦牛心肌组织发育学特点,本实验选择同一海拔梯度不同发育期(妊娠4月龄、出生1日龄、1月龄、3月龄、6龄月)大通牦牛作为研究对象,应用透射电镜技术对不同发育期牦牛心肌细胞线粒体立体形态学指标进行定性和定量分析;运用分子生物学技术检测心肌线粒体标志性酶:细胞色素C氧化酶(Cytochrome C Oxidase,COX)、琥珀酸脱氢酶(Succinatedehydro-genase,SDH)以及低氧诱导因子(Hypoxia-inducible factor,HIF-1α)m RNA进行实时定量荧光分析;应用免疫组织化学方法对牦牛心肌组织血管内皮因子(vascular endothelial growth factor,VEG)和微血管面密度(Microvessel density,MVD)进行检测。研究结果如下:(1)心肌线粒体形态学结果:心肌细胞内线粒体排列整齐均匀,大多呈圆形或卵圆形;外膜、内膜完整,膜间隙、嵴和基质结构清晰可见,由于分娩及外界环境的应激刺激出生1日龄牦牛心肌细胞线粒体结构膨大、数量减少、部分空泡化;心肌线粒体平均截面积、平均体积、面数密度、体积密度随年龄的增加呈上升趋势,差异显著(P0.05);出生1日龄各项指标变化明显,线粒体平均截面积(AX/μm2)、平均体积(V/μm3)均大于其他年龄段,而面数密度(Na/μm2)均低于其他年龄组,体积密度(V/μm3)除大于1月龄均小于其他年龄段。(2)心肌组织COX亚基m RNA表达量结果:出生1日龄时牦牛心肌线粒体COX-I、II m RNA表达量最高;COX-II m RNA表达量均高于COX-Im RNA。(3)心肌组织SDH亚基m RNA表达量结果:心肌组织中SDH 4个亚基转录在出生1日龄SDH 4个亚基表达量均高于其他年龄段。(4)心肌组织HIF-1αm RNA表达量结果:HIF-1αm RNA表达量随年龄增长表达量稳定增加,不同年龄组间差异显著(P0.05);出生1日龄和6月龄HIF-1αm RNA表达量显著高于其他年龄组,差异极显著(P0.01)。(5)心肌组织VEGF、MVD测定结果:不同发育期大通牦牛心肌中VEGF、MVD均有分布,心肌VEGF、MVD百分比含量随年龄的增长而增加,差异显著(P0.05);出生1日龄VEGF、MVD占单位数量心肌纤维的比值较其他年龄组含量低。综上所述,高原低氧环境下,大通牦牛心肌细胞线粒体数目、体积以及心肌组织SDH、COX、HIF-1αm RNA的表达量、心肌组织VEGF、MVD蛋白百分比含量均随年龄增长呈稳定上升趋势。可能由于分娩及外界环境的应激刺激,出生1日龄各项参数与其他年龄组相比均有明显变化,线粒体结构受到暂时性损伤,出生1日龄牦牛在分娩应激刺激下,增加心肌组织HIF-1α表达量和加强调控线粒体标志性酶COX、SDH氧化磷酸化作用使组织细胞快速复氧增加,从而对心肌组织的供氧供能起到一定的保护作用。HIF-1α在细胞核内快速积累,进而诱导机体内VEGF、MVD因子表达调节氧分压防止低氧对细胞造成损伤。
[Abstract]:In order to study the characteristics of myocardial tissue development in Datong yak at different developmental stages under high altitude hypoxia, we selected the same altitude gradient for different developmental periods (4 months of pregnancy, 1 month of birth, 1 month of birth, 3 months of age). Datong yak was used as the research object. The qualitative and quantitative analysis of mitochondrial stereomorphology of cardiac myocytes of yak at different stages of development was carried out by transmission electron microscopy (TEM). Detection of Myocardial Mitochondrial iconic enzymes (cytochrome C Oxidase (Cytochrome C Oxidase,COX), succinate dehydrogenase (Succinatedehydro-genase,SDH) and hypoxia Induction Factor (Hypoxia-inducible factor,) by Molecular Biological techniques HIF-1 伪) m RNA was used for real-time quantitative fluorescence analysis. Vascular endothelial factor (vascular endothelial growth factor,VEG) and microvessel surface density (Microvessel density,MVD) were measured by immunohistochemical method. The results are as follows: (1) the morphology of myocardial mitochondria: the mitochondria in myocardial cells are arranged neatly and evenly, most of them are round or oval; The outer membrane, intima intact, membrane space, cristae and matrix structure were clearly visible. Due to the stress of labor and external environment, the mitochondrial structure of 1 day old yak cardiomyocytes was enlarged, the number of mitochondria was reduced, and part of vacuolation was observed. The mean sectional area, mean volume, face number density and volume density of myocardial mitochondria increased with age (P0.05). At 1 day of birth, the changes of the indexes were obvious. The average mitochondrial area (AX/ 渭 m 2) and mean volume (V / 渭 m 3) were higher than those of other age groups, but the Na/ 渭 m 2 was lower than that of other age groups. The volume density (V / 渭 m 3) was lower than that of other age groups except over 1 month. (2) the expression of COX subunit m RNA in myocardium was the highest in yak myocardial mitochondria at 1 day of life. The expression of COX-II m RNA was higher than that of COX-Im RNA. (3) the expression of SDH subunit m RNA in myocardial tissue was higher than that of other age groups. 4) the expression of HIF-1 伪 m RNA in myocardium: the expression of HIF-1 伪 m RNA increased steadily with age. There were significant differences among different age groups (P0.05). The expression of HIF-1 伪 m RNA at 1 day and 6 months of birth was significantly higher than that of other age groups, and the difference was very significant (P0.01). (5). The results of VEGF,MVD analysis showed that there was a distribution of VEGF,MVD in myocardium of Datong yak at different developmental stages, and the expression of VEGF,MVD in myocardium of Datong yak was significantly higher than that of other age groups. The percentage of MVD increased with age (P0.05). The ratio of VEGF,MVD per unit number of myocardial fibers at 1 day old was lower than that in other age groups. In conclusion, the number and volume of mitochondria, the expression of SDH,COX,HIF-1 伪 m RNA and the percentage of VEGF,MVD protein in myocardium of Datong yak increased steadily with age under high altitude hypoxia. It is possible that the parameters of 1 day old of birth are obviously changed compared with other age groups due to the stress stimulation of labor and external environment. The mitochondrial structure is temporarily damaged, and the 1 day old yak is stimulated by labor stress. Increasing the expression of HIF-1 伪 in myocardium and regulating the oxidative phosphorylation of mitochondrial iconic enzyme COX,SDH increased the rapid reoxygenation of tissue cells. HIF-1 伪 accumulates rapidly in the nucleus, and then induces the expression of VEGF,MVD factor to regulate the partial pressure of oxygen to prevent the damage to cells caused by hypoxia.
【学位授予单位】：青海大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S823.85

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