低分子肝素钙对小鼠胚胎着床的干预作用及机制研究

Effect and mechanism of low molecular weight heparin calcium on mouse embryo implantation

目的探讨低分子肝素钙(Low molecular weight heparin calcium,LMWHC)对小鼠胚胎着床的干预作用及机制。方法构建小鼠妊娠模型,腹腔注射LMWHC干预,观察LMWHC对小鼠胚胎着床的影响。分离培养小鼠滋养层细胞,用不同浓度LMWHC干预细胞,采用Transwell小室检测细胞侵袭力,采用ELISA、Western blot和RT-PCR法检测细胞上清基质金属蛋白酶-2 (matrix metalloproteinases-2,MMP-2)和金属蛋白酶抑制因子-2(tissue inhibitor of metalloproteinase-2,TIMP-2)的蛋白和mRNA水平的表达情况。结果妊娠小鼠腹腔注射LMWHC后,妊娠D6和D9的小鼠胚胎着床数明显增多(P <0. 05);用1×102IU/L、1×103IU/L和1×104IU/L浓度的LMWHC干预滋养层细胞24 h后,滋养层细胞侵袭细胞数分别为(84. 83±14. 15)个、(162. 48±25. 49)个、(98. 73±12. 77)个,与未用LMWHC干预细胞的(65. 27±13. 92)个比较,差异有显著性(P <0. 05),滋养层细胞上清液中MMP-2表达水平较未用LMWHC干预的细胞均升高(P <0. 05),滋养层细胞上清液中TIMP-2表达水平较未用LMWHC干预的细胞降低(P <0. 05)。结论 LMWHC可促进小鼠胚胎着床,其机制可能与LMWHC通过影响MMP-2和TIMP-2表达而增强滋养层细胞侵袭力有关。

Objective To explore the effect and mechanism of low molecular weight heparin calcium(LMWHC)on mouse embryo implantation.Methods A mouse pregnancy model was established and LMWC was injected intraperitoneally.The effects of LMWHC on mouse embryo implantation were then observed.Mouse trophoblasts were isolated,cultured,and treated with various concentrations of LMWHC.Transwell chambers were used to examine cell invasiveness.ELISAs,western blotting,and RT-PCR was used to detect the expression levels of matrix metalloproteinase-2(MMP-2)and tissue inhibitor of metalloproteinase-2(TIMP-2).Results After intraperitoneal injection of LMWHC into pregnant mice,the numbers of embryo implantations at days6and9of pregnancy were increased significantly(P<0.05).At24h after LMWHC injection at1×10^2,1×10^3,and1×10^4IU/L,invading trophoblast numbers were(84.83±14.15),(162.48±25.49),and(98.73±12.77)respectively,significantly different from the cells without LMWHC treatment(65.27±13.92)(P<0.05).The level of MMP-2in the culture supernatant of trophoblasts was higher than that in the culture supernatant of cells without LMWHC treatment(P<0.05),while the level of TIMP-2was lower than that in cells without LMWHC treatment(P<0.05).Conclusions LMWHC promotes mouse embryo implantation,and its mechanism may be related to enhancement of the invasiveness of trophoblasts by affecting the expression of MMP-2and TIMP-2.