缺氧诱导因子1和缺氧诱导因子2在人类着床前胚胎的表达

Expression of hypoxia inducible factor-1 and hypoxia inducible factor-2 in human preimplantation embryos

目的研究缺氧诱导因子1(HIF-1)和缺氧诱导因子2(HIF-2)在人类着床前胚胎各个阶段的表达,探讨这两个氧调节基因在人类早期胚胎发育过程中的作用和意义。方法收集不育症患者捐赠的胚胎,采用巢式逆转录聚合酶链反应(RT-PCR)和实时荧光定量PCR分别定性和定量在5%和20%O2条件下体外培养的人胚胎的HIF-1α和HIF-2αmRNA。采用免疫荧光染色检测人胚胎的HIF-1α和HIF-2α蛋白。结果巢式RT-PCR分别检测了5%和20%O2体外培养的2、4、6、8细胞胚胎和囊胚发现,所有34个胚胎均表达HIF-1α和HIF-2αmRNA。实时荧光定量PCR5%O2培养的人囊胚HIF-1α与18SrRNA的Ct比值为(1.22±0.05);20%O2培养的人囊胚,这一比值是(1.02±0.07);两者比较差异显著(P<0.05)。人胚胎在体外常氧培养条件下的HIF-1αmRNA水平显著高于低氧培养。5%O2培养的人囊胚HIF-2α与18SrRNA的Ct的比值为(1.29±0.04);20%O2培养的人囊胚,这一比值是(1.19±0.11);两者比较无显著差异(P>0.05)。人胚胎在体外低氧培养条件下的HIF-2αmRNA水平与常氧培养无显著差异。5%和20%O2体外培养的2、4、6、8细胞,各个发育阶段人胚胎的HIF-1α和HIF-2α免疫荧光染色均阳性。结论研究结果显示人类着床前胚胎在体外常氧和低氧培养时均表达HIF-1α和HIF-2α。二者可能通过广泛的靶基因系统参与早期胚胎的生长发育和着床过程,在早期胚胎的调控可能不在转录水平,而在转录后水平。

Objective： In early development stage, embryos are exposed to a physiologically low oxygen environment. Both hypoxia inducible factor-1 （HIF-1） and hypoxia inducible factor-2 （HIF-2） are important transcriptional factors that modulate adaptation of cells to hypoxia. In the present study, the expression of HIF-la and HIF-2α at different stages of human preimplantation embryos was investigated. Methods： Human embryos were donated with informed consent by couples attending the Oxford Fertility Unit, John Radcliffe Hospital, Oxford. Ethics approval for the study was obtained from the Central Oxford Research Ethics Committee and the Human Fertilization and Embryology Authority. The nested RT-PCR and real time PCR were used to detect HIF-1α and HIF-2α mRNA in human preimplantation embryos. HIF-la and HIF-2a proteins were detected by immunofluorescence staining. Results： The message RNA of HIF-la and HIF-2α in thirty-four 2-, 4-, 6-and 8-cell human embryos as well as blastocysts cultured at either 5%O2 or 20%O2 conditions were detected by nested RT-PCR. All of these tested embryos were found presence of HIF-1α and HIF-2α mRNA. Their mRNA levels in blastocysts cultured at 5%O2 were further quantified and compared with those at 20%O2 using real time PCR.The Ct value ratio of HIF-1α to 18S rRNA in blastocysts cultured at 5%O2 was 1.22±0.05, and was significantly higher than that at 20%O2 （P〈0.05）. The latter was 1.02±0.07. This result indicated that HIF-1α mRNA level in human blastocysts exposed to hypoxia was significantly lower than that of exposed to normoxia. The Ct value ratio of HIF-2α to 18S rRNA was 1.29±0.04 in blastocysts cultured at 5%O2 and 1.19±0. 11 in that at 20%O2. No significant difference was found between 5%O2 and 20%O2 culture condition. It indicated that HIF-2a mRNA level was comparable in 5 %O2 and 20%O2 cultured human blastocysts. The proteins of HIF-la and HIF-2a in 5% and 20% 02 cultured human embryos were detected by immunofluorescence staining. HIF-la and HIF-2a staining were positive in all of tested 2-, 4-, 6-, 8-cell stage human embryos and blastocysts cultured either 5 %O2 or 20%O2. Conclusions： The present study suggest that human preimplantation embryos express HIF-1α and HIF-2α in vitro under either hypoxia or normoxia conditions. These two molecules may play roles in devel opment and implantation of human embryos via their target genes. It seems that the regulation of HIF-1α and HIF-2α is not at transcriptional level, but possibly at post transcriptional level.