Department of Natural Sciences, Middlesex University, London, United Kingdom.
Eur J Obstet Gynecol Reprod Biol. 2012 Jul;163(1):11-6. doi: 10.1016/j.ejogrb.2012.03.013. Epub 2012 Apr 8.
This study aimed to determine the effects of insulin-like growth factors (IGF-I and IGF-II), heparin, aspirin and vitamin C on the proliferation and apoptosis of human villous cytotrophoblast from first trimester and term placentae.
Villous cytotrophoblast cells were isolated from uncomplicated first trimester (n=12) and term placental tissues (n=12) using negative immunoselection with an antibody to HLA class I antigens. Cells were incubated with IGF-I, IGF-II, heparin, aspirin and vitamin C either alone, or in combination with either TNF-α/IFN-γ or staurosporine. Proliferation was determined by measurement of Ki67 expression using immunocytochemistry. Trophoblast apoptosis was determined by TUNEL staining. Finally RT-PCR was carried out to identify IGF-binding insulin receptor isoforms. Data were expressed as means±SEM. One way analysis of variance (ANOVA) with Bonferroni correction was used to determine if differences between groups were statistically significant.
Following negative immunoselection >98% of cells were positively stained for cytokeratin 7, a marker for cytotrophoblasts, and <1% were vimentin positive. First trimester and term trophoblasts underwent spontaneous apoptosis which was inhibited by approximately 50% in the presence of IGF-II or heparin. Apoptosis was significantly increased following incubation with a combination of TNF-α and IFN-γ or staurosporine. Apoptosis was decreased to basal levels following coincubation with IGF-II or heparin. Incubation with IGFs or heparin resulted in a small, but significant increase in Ki67 expression. Insulin receptor isoform A, which binds IGF-II with high affinity, was present in all trophoblast samples tested.
These results suggest that heparin and IGF-II, but not IGF-I are important regulators of villous cytotrophoblast survival in early and late pregnancy.
本研究旨在确定胰岛素样生长因子（IGF-I 和 IGF-II）、肝素、阿司匹林和维生素 C 对来自早孕期和足月胎盘的人绒毛滋养层细胞增殖和凋亡的影响。
使用针对 HLA Ⅰ类抗原的抗体进行负免疫选择，从正常的早孕期（n=12）和足月胎盘组织（n=12）中分离绒毛滋养层细胞。将细胞与 IGF-I、IGF-II、肝素、阿司匹林和维生素 C 单独或联合 TNF-α/IFN-γ 或 staurosporine 孵育。通过免疫细胞化学测定 Ki67 表达来确定增殖。通过 TUNEL 染色测定滋养层细胞凋亡。最后进行 RT-PCR 以鉴定 IGF 结合胰岛素受体同工型。数据表示为平均值±SEM。采用单因素方差分析（ANOVA）和 Bonferroni 校正来确定组间差异是否具有统计学意义。
经过负免疫选择，>98%的细胞对细胞角蛋白 7（一种滋养层细胞的标志物）呈阳性染色，而<1%的细胞对波形蛋白呈阳性染色。早孕期和足月期滋养层细胞会发生自发凋亡，而 IGF-II 或肝素的存在可抑制约 50%的凋亡。孵育 TNF-α 和 IFN-γ 或 staurosporine 后，凋亡显著增加。与 IGF-II 或肝素共同孵育后，凋亡降至基础水平。IGF 或肝素孵育可导致 Ki67 表达略有增加，但具有统计学意义。胰岛素受体同工型 A 存在于所有测试的滋养层样本中，其与 IGF-II 具有高亲和力。
这些结果表明，肝素和 IGF-II（而非 IGF-I）是早孕期和晚期妊娠绒毛滋养层细胞存活的重要调节因子。
