高原鼢鼠血管内皮生长因子基因编码和mRNA的表达以及微血管密度:与其它鼠类的比较

Gene coding and mRNA expression of vascular endothelial growth factor as well as microvessel density in brain of plateau zokor:comparison with other rodents

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。为进一步探讨高原鼢鼠对严重低氧、高CO2洞道环境的适应机制,本文就高原鼢鼠脑组织中血管内皮生长因子(vascular endothelial growth factor,VEGF)的mRNA表达水平及MVD与其它鼠类进行了比较研究。提取高原鼢鼠肝组织中总RNA,应用RT-PCR方法获得VEGF cDNA,并对VEGF基因进行克隆、测序,获得VEGF基因编码区;从GenBank获得高原鼠兔(Ochotona curzniae)、大鼠(Rattus norvegi-cus)和小鼠(Mus musculus)VEGF cDNA编码区碱基序列,应用生物学分析软件对高原鼢鼠、高原鼠兔、大鼠和小鼠VEGF cDNA及其所编码蛋白的氨基酸同源性进行了比较;通过应用SYBR GreenΙ荧光定量法和免疫组织化学方法分别对高原鼢鼠、高原鼠兔和Sprague-Dawley(SD)大鼠脑组织中VEGF mRNA的表达水平及脑MVD进行测定。结果显示,高原鼢鼠VEGF基因的编码区为645bp,与高原鼠兔、大鼠和小鼠VEGF基因的同源性分别为92.1%、93.6%和93.8%。高原鼢鼠VEGF基因编码188个氨基酸,其中1～26氨基酸为信号肽。高原鼢鼠VEGF188与高原鼠兔VEGF189、大鼠和小鼠VEGF188氨基酸同源性分别为90.2%、94.9%和94.4%。高原鼢鼠脑组织中VEGF基因mRNA表达水平显著低于SD大鼠(P<0.05),而与高原鼠兔没有明显差异(P>0.05);高原鼢鼠、高原鼠兔、SD大鼠脑组织中MVD依次减小,且差异显著(P<0.05)。综上所述,高原鼢鼠通过提高脑组织中MVD增强对洞道低氧环境的适应能力,但是高原鼢鼠脑组织中VEGF基因mRNA表达水平相对SD大鼠较低,可能与其洞道中高浓度CO2对VEGF基因表达的抑制作用有关。

Vascular endothelial growth factor（VEGF） plays an important role in tissues angiogenesis.The adaptation of animals to hypoxic environment is relative to the microvessel density（MVD） in tissues.To further explore the adaptation mechanisms of plateau zokor（Myospalax baileyi） to the hypoxic-hypercapnic burrows,the VEGF mRNA and the MVD in cerebral tissues of the plateau zokor were studied.Total RNA was isolated from liver,and VEGF cDNA was obtained by RT-PCR,then the VEGF cDNA was cloned and sequenced.The coding sequence of plateau pika（Ochotona curzniae）,rat（Rattus norvegicus） and mouse（Mus musculus） VEGF cDNA are obtained from GenBank,and the nucleotide and amino acid sequence homology of plateau zokor VEGF cDNA coding sequence with that of plateau pika,rat and mouse were analyzed and compared by using of bioinformatics software.The VEGF mRNA was detected by real-time PCR,and the MVDs in cerebral tissues of the plateau zokor,plateau pika and Sprague-Dawley（SD） rat were measured by immunohistochemical staining.The results showed that the open reading frame of the plateau zokor VEGF was 645 bp,and 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 was 90.2%,94.9% and 94.4% homologous to that of plateau pika,rat and mouse.The level of VEGF mRNA in brain of the plateau zokor was significantly lower than that of SD rat,but there was no obvious difference in VEGF mRNA level between plateau zokor and plateau pika.The MVD in brain of the plateau zokor was markedly higher than that of plateau pika and SD rat.In conclusion,plateau zokor enhances its adaptation to the hypoxic environment by increasing the MVD.The level of VEGF mRNA in the brain of plateau zokor is lower than that of SD rat,which may be as a result of inhibition by the higher concentration of carbon dioxide in the burrow.