ISG15和OAS1在奶牛早期妊娠阶段外周血中的表达规律

Study on ISG15 and OAS1 Transcriptions in Peripheral Blood of Dairy Cows during Early Pregnancy

本研究旨在揭示干扰素刺激基因ISG15和OAS1在中国荷斯坦奶牛早期妊娠阶段外周血中的表达规律,为利用干扰素刺激基因进行早期妊娠诊断提供理论和方法上的指导。试验用青年母牛共27头,其中自然发情牛10头,同期发情牛17头;人工授精后0、14、18、21和28d采集外周血并分离总白细胞或白细胞亚类(淋巴细胞、单核细胞、NK细胞);采用荧光定量PCR检测ISG15和OAS1的相对表达量。结果表明:人工授精后18d妊娠牛外周血中ISG15和OAS1的相对表达量均显著高于空怀牛;ISG15在妊娠牛中一致上调(平均3.92倍),空怀牛中一致下调(平均1.46倍);OAS1在妊娠牛中平均上调3.49倍,空怀牛平均下调1.28倍。自然发情条件下,妊娠牛外周血中ISG15和OAS1的相对表达量均显著高于空怀牛,同期发情条件下两个基因在表达趋势上与之类似,但差异不显著;进一步分析显示:人工授精后18dOAS1在妊娠牛淋巴细胞中的表达显著高于空怀牛,其中妊娠牛中OAS1一致上调表达,平均上调2.14倍,空怀牛中OAS1一致下调,平均下调1.80倍。研究提示:人工授精后第18天妊娠牛与空怀牛之间ISG15的差异表达比OAS1更明显;自然发情状态下,妊娠牛与空怀牛之间两个基因差异表达情况比同期发情状态下更明显;相对于外周血总白细胞,妊娠牛与空怀牛外周血淋巴细胞中ISG15、OAS1的差异表达倍数更低。综上表明,利用外周血中ISG15的表达水平可望能够在人工授精后18d对自然发情奶牛较为准确地做出早期妊娠诊断;对于ISG15、OAS1在外周血免疫细胞亚类中的表达分析,为深入研究奶牛早期妊娠识别的分子机制奠定了基础。

This study aimed to investigate mRNA transcriptions of interferon stimulated genes ISG15 and OAS1in the peripheral blood of Chinese Holstein dairy cows during early pregnancy,by which we hope to provide an insight into their applications in bovine early pregnancy diagnosis.A total of 27 heifers following spontaneous estrus(n=10)or oestrus synchronisation(n=17)were used for the study.Peripheral blood were collected at 0,14,18,21 and 28dpost artificial insemination(AI);subsets of peripheral blood leukocytes(lymphocyte,monocyte,and natural killer cell)were isolated at 0,18 and 21d;Real-time PCR was employed to detect the relative mRNA transcriptions of ISG15 and OAS1in the whole white blood cells(WBC)and/or the leukocytesubpopulations.The results showed that bovine ISG15 and OAS1in the peripheral blood of pregnant cows were significantly elevated versus non-pregnant cows at 18d(P<0.05);On 18 dpost AI,ISG15 was up-regulated in pregnant cows with the average fold change(FC)of 3.92,whereas it was down-regulated in non-pregnant cows with the average FC of 1.46;OAS1was similarly upregulated(FC=3.49)in pregnant cows and down-regulated(FC=1.28)in non-pregnant cows.The relative transcriptions of ISG15 and OAS1in pregnant cows following spontaneous estrus were significantly higher than those in non-pregnant cows at 18 dpost AI;although the tendency in cows following oestrus synchronisation was the same,the difference was not statistically significant.Further analysis in the subpopulations of leukocytes showed that OAS1 in lymphocytes of pregnant cows was significantly elevated(+2.14),whereas depressed in non-pregnant cows(-1.80)at 18 dpost AI(P<0.05).The results indicated that regulations of ISG15 in peripheral blood of pregnant cows were more obvious than OAS1 at 18dafter AI;differential expressions of ISG15 and OAS1between pregnant cows and non-pregnant cows were more obvious in cows following spontaneous estrus.In conclusion,ISG15 transcriptions in peripheral WBC could be a better indicator for bovine early pregnancy diagnosis(18dpost AI),especially in cows following spontaneous estrus.Gene expression analysis of bovine ISG15 and OAS1in the leukocyte subsets has provided additional insight into their roles during early maternal recognition of pregnancy.