缺氧诱导因子1α在缺氧造成子宫内膜容受性低下中的作用

摘要

目的 探讨缺氧诱导因子1α(HIF-1α)在缺氧造成子宫内膜容受性低下的作用机制.方法 CoCl2 所致低氧环境下培养RL95-2细胞,采用逆转录PCR技术及蛋白印迹法检测HIF-1α及肿瘤坏死因子样凋亡的微弱诱导因子(TWEAK)mRNA和蛋白的表达水平,流式细胞仪分析细胞凋亡率;并在复发性流产(RSA)妇女的子宫内膜组织中,采用免疫组化方法 检测HIF-1α、TWEAK的表达,透射电镜观察炎性反应细胞及细胞凋亡的超微形态,以男方因素的不孕妇女作为对照.结果 (1)低氧环境下培养不同时间(0、12、24、48 h),RL95-2细胞中HIF-1α mRNA的表达水平依次为0.272±0.010、0.354 ±0.020、0.591±0.020、0.890±0.020,TWEAK mRNA的表达水平依次为:0.104±0.010、0.510±0.020、1.021±0.020、1.237±0.040;两个因子12、24、48 h与0 h之间分别比较,差异均有统计学意义(P＜0.05).(2)各时间点HIF-1α、TWEAK蛋白的表达,HIF-1α的表达水平依次为0.853±0.010、0.931±0.030、1.124±0.010、1.317±0.020,TWEAK的表达水平分别为0.042±0.010、0.091±0.010、0.131±0.020、0.205±0.030;两种蛋白的表达在12、24、48 h与0 h之间分别比较,差异均有统计学意义(P＜0.05),同时24、48 h与12 h之间比较,差异也有统计学意义(P＜0.05).(3)随着缺氧时间的延长,细胞凋亡率明显增加,各时间点细胞凋亡率分别为(3.2±1.4)%、(16.2±3.2)%、(26.3 ±3.5)%、(31.8±3.5)%;12、24、48 h与0 h之间分别比较,差异均有统计学意义(P＜0.05).(4)在RSA妇女的子宫内膜上皮细胞及间质细胞中,HIF-1α的阳性率分别为32.3%、8.4%.对照妇女为16.7%、7.3%;TWEAK的阳性率分别为28.3%、3.9%,对照妇女为11.6%、2.7%;两者之间两个因子分别比较,差异均有统计学意义(P＜0.05).电镜观察到RSA妇女的子宫内膜上皮及间质炎性细胞浸润及细胞凋亡明显.结论 HIF-1α、TWEAK参与缺氧导致的子宫内膜上皮细胞炎性反应及凋亡,可能参与缺氧造成子宫内膜容受性低下的作用机制,HIF-1α有可能作为新的治疗靶点改善子宫内膜容受性低下.%Objective To study the mechanism of hypoxia inducing factor-1α(HIF-1α)pathway in establishment of hypoxia inducing low endometrial receptivity.Methods RL95-2 cell lines.the ideal model of study ER,were cultured in hypoxia condition induced by CoCl2,and the expression of mRNA and protein of HIF-1α and tumor necrosis factor like weak inducer of apoptosis(TWEAK)were measured by reverse transcription-PCR and western blot. The apoptosis rate was analyzed by flow eytometry.Then the mechanism confirmed by comparing the two factors in endometrium and the ultra-appearance of inflammatory reaction and apoptosis between recurrent spontaneous abortion women and control women.Results (1)On difierent time point(0,12,24,48 hour),mRNA expression of HIF-1α were 0.272±0.010,0.354±0.020,0.591±0.020.0.890±0.020,while the expression of TWEAK were 0.104±0.010,0.510±0.020,1.021±0. 020, 1. 237 +0. 040, respectively, the expression level between 12, 24, 48 and 0 hour all showed significant differences (P＜0. 05 ). (2) Protein expression of HIF-1α were 0. 853 +0. 010, 0. 931 ±0. 030,1. 124±0.010, 1.317±0.0 20 respectively, while was 0.042±0.010, 0.091 ±0.010, 0. 131±0.020,0. 205 ±0. 030 in TWEAK expression, the different level were statistically significant ( P＜0. 05 ). ( 3 )With longer culture under hypoxia, the cell apoptosis rate increased obviously. The apoptosis rate of each time point were ( 3.2±1.4 ) %, ( 16. 2 ±3.2 ) %, ( 26. 3±3.5 ) %, ( 31.8±3.5 )%, the differences between 12, 24, 48 and 0 hour had significance (P ＜0. 05). (4) The positive rate of HIF-1α stained in epithelium cells and stroma cells of test group were 32. 3%, 8.4% and 16. 7%, 7. 3% in control group. The positive rate of TWEAK were 28. 3%, 3.9% in recurrent spontaneous abortion group and 11.6%, 2. 7% in control group ( P ＜0. 05 ). The ultra-appearance of inflammatory cell infiltrated and apoptosis were obvious in test group. Conclusions Cell inflammation reaction and apoptosis induced by HIF-1α pathway may participate the mechanism of hypoxia inducing low endometrial receptivity. HIF-1α might become a novel target for improving poor endometrial receptivity.