【作者】 郑亚宁；

【导师】 魏登邦；

【作者基本信息】 青海大学 ， 基础兽医学， 2011， 硕士

【摘要】 高原鼢鼠（Myospalax bailey）是青藏高原特有的物种之一,血管内皮生长因子（Vascular endothelial growth factor, VEGF）具有促进毛细血管增生的功能,动物组织微血管密度（Microvessel density, MVD）的大小与其对低氧的适应能力有关。为了研究高原鼢鼠对高原环境的适应机制,通过分子克隆及生物信息学方法测定并分析了高原鼢鼠VEGF的cDNA序列。根据VEGF基因结构特点,设计特异性表达VEGF各亚型的探针,应用Real-time PCR测定不同海拔高原鼢鼠骨骼肌内VEGF各亚型mRNA表达水平。通过免疫组织化学方法对不同海拔高原鼢鼠骨骼肌微血管密度进行测定,并运用SPSS17.0软件对MVD和各VEGF亚型基因表达量之间进行相关性分析。结果:获得的VEGF编码区为645bp,其序列与高原鼠兔VEGF₁₈₉、大鼠和小鼠VEGF₁₈₈cDNA序列的同源性分别为92.1%、93.6%和93.8%。该序列编码蛋白长度为214个氨基酸,在N端包含一个26个氨基酸的信号肽,成熟的蛋白具有188个氨基酸,为VEGF₁₈₈亚型。高原鼢鼠VEGF₁₈₈与高原鼠兔VEGF₁₈₉、大鼠和小鼠VEGF₁₈₈的同源性分别为90.2%、94.9%和94.4%。高海拔高原鼢鼠骨骼肌MVD（1107.47±47.98）显著高于低海拔高原鼢鼠（918.74±54.10）（P<0.05）。高原鼢鼠骨骼肌VEGF亚型主要组成为VEGF₁₈₈、VEGF₁₆₄和VEGF₁₂₀。4200m海拔的高原鼢鼠骨骼肌中总量VEGF、VEGF₁₈₈、VEGF₁₆₄ mRNA表达量（分别为1.27±0.24,1.22±0.13,1.47±0.24）显著高于3200m海拔高原鼢鼠表达量（分别为0.90±0.10,0.66±0.14,0.94±0.17）（P<0.05）,VEGF₁₂₀ mRNA表达量（4200m海拔为0.58±0.11,3200m海拔为0.67±0.12）没有显著变化。VEGF₁₆₄与MVD显著相关（P<0.05）。以上结果表明,高原鼢鼠骨骼肌VEGF亚型组成以VEGF₁₆₄为主,VEGF₁₆₄基因是促进毛细血管增生作用的主要形式。VEGF₁₈₈、VEGF₁₆₄和VEGF₁₂₀对低氧的敏感程度不同,他们的对低氧敏感程度依次降低。更多还原

【Abstract】 Plateau zokor （Myospalax bailey） is native to the Qinghai-Tibet plateau, vascular endothelial growth factor （VEGF） can promote capillary proliferation, and the microvessel density （MVD） of animal tissues is related to their adaptility to hypoxia. To study their adaptive mechanisms, the VEGF cDNA of plateau zokor was sequenced and analyzed by bioinformatics, and the expression patterns of VEGF in skeletal muscle tissues were measured through real-time PCR and the MVD of skeletal muscle of plateau zokor in different altitudes were measured by immunohistochemical staining and the correlation relation of MVD with VEGF expression was analyzed by SPSS17.0. Our results indicated that, the open reading frame of the plateau zokor VEGF was 645 bp, the coding sequence of the plateau zokor VEGF cDNA shared 92.1%, 93.6% and 93.8% nucleotide sequence homology to that of the plateau pika, rat and mouse, respectively. The deduced amino acid sequence of the plateau zokor VEGF cDNA was composed of 188 amino acids and the amino acids from 1 to 26 were signal peptide sequence. The plateau zokor VEGF₁₈₈ shared 90.2%、94.9% and 94.4% homologous to that of the plateau pika, rat and mouse, respectively. MVD （1107.47±47.98） in high altitude plateau zokor was significantly higher than that （918.74±54.10） of skeletal muscle in low altitude plateau zokor （P<0.05）. VEGF isoforms in plateau zokor skeletal muscle is mainly composed of VEGF₁₈₈, VEGF₁₆₄ and VEGF₁₂₀. The expression of total VEGF, VEGF₁₈₈, VEGF₁₆₄ mRNA （respectively 0.90±0.10, 0.66±0.14, 0.94±0.17） in plateau zokor at low altitude was significantly lower than high altitude platau （1.27±0.24, 1.22±0.13, 1.47±0.24 respectively）（P<0.05）, while VEGF₁₂₀ mRNA expression did not change significantly（0.67±0.12 at low altitude, 0.58±0.11 at high altitude）. VEGF₁₆₄ was significantly correlated with MVD （P <0.05）.In conclusion, VEGF main isoform in plateau zokor skeletal muscle is VEGF₁₆₄, VEGF₁₆₄ is the main form to advance capillary proliferation in VEGF forms.The sensitivities to hypoxia of VEGF₁₈₈, VEGF₁₆₄ and VEGF₁₂₀ were different, and in turn reduced.更多还原