缺氧诱导因子-1在弓形虫感染孕鼠中的表达

Expression of hypoxia-inducible factor-1 in mice infected with Toxoplasma gondii during pregnancy

目的探讨缺氧诱导因子-1(hypoxia-inducible factor-1,HIF-1)在小鼠妊娠早期感染弓形虫后母胎界面的表达及可能发挥的作用。方法将20只妊娠C57BL/6小鼠(体质量为16~20 g)随机分为4组,即12 d对照组、12 d感染组和18 d对照组、18 d感染组。在小鼠孕6 d时,给予12 d感染组、18 d感染组小鼠腹腔注射弓形虫PRU株速殖子150个/只,孕12 d、孕18 d对照组孕鼠则注射等量PBS。于孕12、18 d分别处死相应对照组和感染组小鼠,取各组孕鼠胎盘和子宫组织观察其组织病理变化;采用实时荧光定量PCR(q PCR)技术检测孕鼠胎盘和子宫中HIF-1α、HIF-1β及血管内皮生长因子(VEGF)m RNA表达水平,并分析HIF-1α和VEGF m RNA表达水平的相关性。此外,设置孕12 d感染组和孕18 d感染组PBS阴性对照组,采用免疫组化染色检测孕鼠胎盘和子宫组织中HIF-1α表达水平。结果与孕12 d、孕18 d对照组相比,孕12 d、孕18 d感染组孕鼠出现畸胎、胎盘发育不良等不良妊娠结局。HE染色结果显示,与相应对照组相比,孕12 d、孕18 d感染组小鼠胎盘组织迷路区细胞分别出现肿胀和瘀血,且均出现血窦减少、炎性细胞浸润;两组小鼠子宫组织均有柱状上皮细胞损伤和炎性细胞浸润。q PCR检测结果显示,与同期对照组相比,孕12 d感染组小鼠胎盘组织中HIF-1α、HIF-1βm RNA表达水平显著上升(F=132.6、286.9,P均<0.05),子宫组织中HIF-1α、HIF-1βm RNA表达水平显著下降(F=111.5、55.2,P均<0.05);孕18 d感染组小鼠胎盘和子宫组织中HIF-1α、HIF-1βm RNA表达水平均显著下降(F=215.8、418.9、156.8、200.1,P均<0.05)。与同期对照组相比,孕12 d感染组小鼠胎盘组织中VEGF-A、VEGF-B和VEGF-C m RNA表达水平均显著下降(F=426.2、104.6、566.9,P均<0.05),子宫组织中VEGF-A、VEGF-B和VEGF-C m RNA表达水平显著上升(F=95.8、502.4、610.0,P均<0.05);孕18 d感染组小鼠胎盘组织和子宫组织中VEGF-A、VEGF-B和VEGF-C m RNA表达水平均显著上升(F=521.9、100.6、275.9、224.6、108.2、333.4,P均<0.05)。免疫组化染色结果显示,与相应对照组相比,孕12 d、孕18 d感染组小鼠胎盘迷路区HIF-1α分别呈强阳性和弱阳性表达,子宫组织中HIF-1α均不表达。结论小鼠孕早期感染弓形虫后,胎盘组织中HIF-1α表达呈现孕中期上升、孕晚期下降的趋势,且与VEGF-A、VEGF-B、VEGF-C表达变化趋势相反;推测HIF-1α通过抑制VEGF表达而抑制小鼠妊娠期间胎盘血管生成,导致不良妊娠结局。

Objective To investigate the expression and possible role of hypoxia-inducible factor-1(HIF-1)at the maternalfetal interface following Toxoplasma gondii infection during early pregnancy.Methods Twenty pregnant C57 BL/6 mice,each weighing 16 to 20 g,were randomly divided into 4 groups,including the 12-d control group,12-d infection group,18-d control group and 18-d infection group.Mice in the 12-d and 18-d infection groups were injected intraperitoneally with 150 tachyzoites of the T.gondii PRU strain on day 6 of pregnancy,while mice in the 12-d control and 18-d control groups were injected with the same volume of phosphate buffered saline(PBS).Mice in the control and infection groups were sacrificed on days 12 and 18 of pregnancy,and the placental and uterine specimens of the pregnant mice in each group were sampled for pathological examinations.The mRNA expression of HIF-1α,HIF-1βand vascular endothelial growth factor(VEGF)was quantified using quantitative fluorescent real-time PCR(qPCR)assay in the placental and uterine specimens,and the correlation between HIF-1αand VEGF mRNA expression was examined.In addition,and the HIF-1αexpression was detected using immunohistochemical staining in the placental and uterine specimens of pregnant mice.Results Compared with the 12-d and 18-d control groups,adverse pregnant outcomes were observed in mice in 12-d and 18-d infection groups,such as teratism and placental dysplasia.HE staining showed swelling and blood stasis of cells,sinusoid reduction and inflammatory cell infiltration in the labyrinth area of the placenta specimens of mice in 12-d and 18-d infection groups relative to 12-d and 18-d control groups,and columnar epithelial cell injury and inflammatory cell infiltration were seen in the mouse uterine specimens in both infection groups.qPCR assay detected significantly higher HIF-1α(F=132.6,P<0.05)and HIF-1βm RNA expression(F=286.9,P<0.05)in the placental specimens and lower HIF-1α(F=111.5,P<0.05)and HIF-1βmRNA expression(F=55.2,P<0.05)in the uterine specimens in the12-d infection group than in the 12-day control group,and significantly lower HIF-1αand HIF-1βmRNA expression was detected in the placental and uterine specimens in the 18-d infection group than in the 18-day control group(F=215.8,418.9,156.8 and 200.1;all P values<0.05).Significantly lower VEGF-A(F=426.2,P<0.05),VEGF-B(F=104.6,P<0.05)and VEGF-C mRNA expression(F=566.9,P<0.05)in the placental specimens and higher VEGF-A(F=426.2,P<0.05),VEGF-B(F=104.6,P<0.05)and VEGF-C mRNA expression(F=566.9,P<0.05)in the uterine specimens were detected in the 12-d infection group than in the 12-d control group,and higher VEGF-A,VEGF-B and VEGF-C m RNA expression was found in the placental and uterine specimens in the 18-d infection group than in the 18-d control group(F=521.9,100.6,275.9,224.6,108.2 and333.4;all P values<0.05).Immunohistochemical staining showed strongly and mildly positive HIF-1αexpression in the mouse placental labyrinth area in the 12-d and 18-d infection groups relative to 12-d and 18-d control groups,while no HIF-1αexpression was detected in mouse uterine specimens.Conclusions HIF-1αexpression appears a tendency towards a rise in the second trimester and a reduction in the third trimester in mice following T.gondii infection during early pregnancy,which is contrary to the changing tendency of VEGF-A,VEGF-B,and VEGF-C expression.It is hypothesized that HIF-1αinhibits placental angiogenesis in mice during pregnancy through suppressing VEGF expression,resulting in adverse pregnant outcomes.