【作者】 郑澜；

【导师】 陆爱云；

【作者基本信息】 上海体育学院 ， 运动人体科学， 2004， 博士

【摘要】 研究目的:通过研究低氧运动对肌组织血管生成低氧反应基因的转录调节、低氧反应基因产物对肌组织血管生成的促进作用、低氧运动肌组织血管的生成机制,来探讨低氧运动促进肌组织血管生成的机制,为低氧训练在运动实践中的运用提供理论依据和应用途径。研究方法:应用低氧细胞培养、凝胶迁移率变动实验方法,研究低氧调节培养人脐静脉内皮细胞HIF-1与VEGF、Flt-1 DNA结合活性。应用3×3析因设计试验,采用递增负荷运动训练方案,及低氧和超低氧两种不同程度的递增低氧刺激,在运动中和运动后给予低氧处理,建立大鼠不同运动方式后进行不同程度低氧刺激动物模型,在此基础上,应用血气分析、免疫组织化学、原位杂交及计算机图像分析等方法,研究低氧运动对大鼠动脉血氧合状态的影响,及动脉血氧合程度的下降对肌组织HIF-1α的影响,进而对低氧运动肌组织VEGF、Flt-1基因转录的促进作用;应用酶联免疫吸附检测、免疫组织化学及计算机图像分析及体视学等方法,研究VEGF、Flt-1对低氧运动肌组织血管生成的促进作用;应用透射电镜研究方法,从形态学角度研究低氧运动肌组织血管生成的方式。结论:采用不同氧含量的混合气体进行脐静脉内皮细胞培养,为低氧训练肌组织血管生成的离体研究提供了低氧细胞培养模型;应用3×3析因设计试验方法,采用递增负荷跑台运动训练和递增低氧程度低氧刺激,建立了不同低氧处理及运动方式的低氧训练动物实验模型。单纯低氧(氧含量由18.2kPa逐周下降至15.2 kPa )或超低氧(氧含量由17.4 kPa逐周下降至11.3kPa )处理,不能增加肌组织微血管密度,常氧训练、低氧训练,以及训练后进行低氧处理,可增加肌组织微血管密度。低氧处理与训练方式两种因素对肌组织微血管密度的增加具有协同交互作用,并且低氧处理、训练方式的主效应有差别。从常氧训练与低氧训练微血管数量变化的差异来看,低氧训练对肌组织微血管的影响优于常氧训练。低氧及运动使心肌、骨骼肌组织中微血管密度增加,有利于运动中肌组织对氧、营养物质的摄取,及代谢产物的排出,表明在运动训练中或训练后施以低氧条件刺激,可作为一种增强机体耐缺氧能力的辅助训练手段。对肌组织毛细血管超微结构的研究发现,低氧运动肌组织血管生成可通过芽生或非芽生的方式进行,这两种血管生成方式中以非芽生方式即套迭式微血管生长方式为主要形式,表明低氧运动可使肌组织在满足能量和代谢方面采用更快、更经济的血管生成方式,促进血管物质交换功能的增强,来提高机体的运动能力。低氧运动血管生成的转录调节机制为:由于离体情况下,低氧可增加培养人脐静脉内皮细胞中HIF-1与血管生成低氧反应基因VEGF、Flt-1 DNA结合活性,这种结合活性在一定氧张力范围内(培养液中氧分压不高于80.50mmHg,此时混合培养气体中的氧含量为7%)受低氧浓度的调节;在体情况下,低氧及运动可降低动脉血氧合程度,低氧训练是使动脉血氧分压下降的最有效刺激;并且低氧运动可使HIF-1α蛋白、VEGF mRNA、Flt-1 mRNA增加。因此受机体氧合程度的下降,低氧运动可增强肌组织中HIF-1α蛋白表达,低氧和运动肌组织HIF-1α蛋白表达的增加可促进VEGF、Flt-1的基因转录。低氧运动促进肌组织血管生成的作用机制为:低氧运动可使肌组织血管生成低氧反应基因VEGF、Flt-1的蛋白产物增加,VEGF蛋白产生后,可通过自分泌或旁分泌的方式,与肌组织中血管内皮细胞膜上的Flt-1受体结合,参与肌组织血管生成;低氧、运动以及低氧运动都能使血清VEGF含量减少,而此时肌组织VEGF蛋白含量增加,由于肌组织Flt-1受体含量增加,而增加了肌组织对循环中VEGF的摄取、利用,减少了血清VEGF含量。因此,血清VEGF含量可作为反映机体耐低氧能力的指标。更多还原

【Abstract】 Objective:In order to provide a theoretical basis and applied methods for hypoxic training applied in sports practice, this thesis studied the mechanism of hypoxic training promoting angiogenesis on muscular tissue by exploring the effect of hypoxic training on regulating the hypoxic responsive genes on muscular issue, the effect of the hypoxic responsive genes on boosting angiogenesis on muscular issues, and the angiogenesis mechanism of hypoxic training muscular tissue.Material and Methods:Hypoxia cell culture and electrophoretic mobility shift assay were applied to study the protein-DNA binding activity of hypoxia induced factor-1 and vascular endothelium growth factor gene, and fms-like tyrosine kinase-1 of human umbilicus vein endothelial cell under hypoxia. 3×3 factorial experiment, progressive treadmill exercise, hypoxia and super-hypoxia increasing by degree were used to establish animal model with different training pattern and different hypoxic stimulus. Thereafter, Blood-gas analysis, in situ hybridization, immunohistochemical technology and computer image processing methods were used to study the effect of hypoxic training on the oxygen binding status of arterial blood, the effect of oxygen binding status of arterial blood on hypoxia induced factor-1αof muscular tissue, and then the promoting effect of hypoxic training on genes transcription of vascular endothelium growth factor and fms-like tyrosine kinase-1. In addition, Enzyme linked immunosorbnent assay, Stereology, immunohistochemical technology and computer image processing methods were used to study the accelerating effect of vascular endothelium growth factor and fms-like tyrosine kinase-1 on hypoxic training angiogenesis of muscular tissue. In the end, transmission electron microscope was applied to study the morphological mode of angiogenesis of hypoxic training muscular tissue.Conclusions:Different Oxygen content mixed gases were administered to culture human umbilical vein endothelium cell to found hypoxia cell culture model for ex vivo study of hypoxic training, and an animal model of hypoxic training was successfully established by progressive treadmill exercise and hypoxic stimulus with progressive hypoxia.Simple hypoxia (hypoxia content from 18.2kPa to 15.2 kPa ) and super- hypoxia (hypoxia content from 17.4 kPa to 11.3kPa ) could not increase density of micro- blood vessel. Normoxic training, hypoxic training, and hypoxic administration after training could increase density of micro- blood vessel. Interaction occurred between hypoxic administration and training pattern, and hypoxic administration and training pattern had different main effects. From the changing of micro- blood vessel, hypoxic training was found to be better than normoxic training to micro- blood vessel on muscular tissue.In vivo, hypoxia could increase binding activity of HIF-1 of culture human umbilical vein endothelium cell and VEGF、Flt-1 DNA. The binding activity was regulated by oxygen content within a certain range. Ex vivo, hypoxia could decrease arterial blood oxygen binding; hypoxic training was the most efficient stimulus to decrease arterial partial pressure of oxygen, while it could decrease arterial blood oxygen binding to a large degree. The transcription regulation mechanism of angiogenesis of hypoxic training muscular tissue was: affected by the degree of oxygen binding, hypoxic training could increase the protein expression of HIF-1 o?n muscular tissue and the increase could promote the genes transcription of VEGF and Flt-1.The mechanism of hypoxia responsive genes promoting the angiogenesis of hypoxic training muscular tissue was: hypoxic training could increase the protein of angiogenesis hypoxia responsive genes VEGF and Flt-1, and after VEGF protein was produced, it could secrete by autocrine or by paracrine, combine with Flt-1 receptor on the vascular endothelium cell membrane, and participate in the angiogenesis of muscular tissue. Hypoxia, training, and hypoxic training all could reduce the content of serum VEGF, meanwhile, the proteins of VEGF on muscular tissue increased and the Flt-1 receptors also increased. Therefore, ingestion and utilization of VEGF from circulation was increased on muscular tissue.Angiogenesis on muscular tissue could be performed by means of sprouting and no-sprouting, among which no-sprouting angiogenesis pattern, i.e. intussusceptive microvascular growth, was the major way, suggesting that muscular tissue could take faster and more economical angiogenesis pattern to satisfy the demands of energy and metabolization.更多还原