凝血酶刺激单核/巨噬细胞产生IL-8增强卵巢癌细胞侵袭力的实验研究

MO/MA stimulated by thrombin enhanced EOC cells invasion potential through IL-8 secretion.

目的:研究凝血酶刺激正常人外周血来源单核/巨噬细胞(MO/MA)后对卵巢癌细胞侵袭力的影响。方法:分离正常女性外周血单核细胞后,用印度墨汁吞噬试验检测凝血酶刺激MO/MA后吞噬功能的变化;以凝血酶刺激MO/MA后的上清为趋化物,检测对卵巢癌细胞系(ES-2,SKOV-3,HO-8910)体外侵袭力的影响;阻断侵袭实验中加入IL-8单克隆中和抗体。转录因子试剂盒AP1、STAT、Inflammation 1、Oncogene 3家族筛选凝血酶通过何种细胞信号转导途径激活MO/MA。结果:(1)凝血酶未显著提高MO/MA的吞噬能力;(2)基质胶体侵袭实验表明,上皮性卵巢癌细胞系ES-2在MO/MA+凝血酶刺激组上清作用下,侵袭力增强(161.9±11.18,n=6),其侵袭细胞数目与卵巢癌腹水刺激组(157.5±14.86,n=4)类似,但显著高于阴性对照组普通培养基(47.25±12.45,P<0.05),添加水蛭素(凝血酶抑制剂)后能显著降低其侵袭力(73.5±17.3,P<0.01);抗IL-8单克隆抗体对ES-2细胞侵袭的阻断作用呈浓度依赖性,相同体外侵袭实验在SKOV-3细胞系(n=2)及HO-8910细胞系(n=2)中得到类似结果;(3)转录因子检测表明,In-flammation 1及Oncogene 3家族中几乎所有涉及炎症的转录因子包括c-Fos,c-Rel,NF-κbp50,NF-κb p65,c/EBPa,Egr-1,HIF-1,OctⅠ,OctⅡ在凝血酶刺激巨噬细胞后活性增加(n=4)。结论:凝血酶刺激MO/MA通过上调IL-8产生,显著增强卵巢癌细胞的侵袭力,表明卵巢癌腹膜内凝血酶可能通过教育和诱导MO/MA向TAM样细胞分化后,加速肿瘤细胞腹膜内转移。

Objective：To explore the impact of coagulation factor Ⅱ（thmmbin） stimu-lating monocytes/macrophages（MO/MA） on epithe]ial ovarian cancer （EOC） invasion potential. Method：India ink was used to detect phagocytic capacity of MO/MA stimulated by thrombin. The supernatant of MO/MA treated by thrombin or thrombin＋hirudin for 12 hours was collected and co-cultured with ovarian cancer cell line ES-2, SKOV-3, HO-8910. The invasive ability of tumor cells was measured. IL-8 monoclonal neutralization antibody was added in blocking experiments. Transfactor kits including AP-1, STAT, inflammation 1 and Oncogene 3 families were used to detect the pathway mediaotors involved in MO/MA activation by thrombin. Resuits： （1）Thrombin didn＇t increase MA phagocytosis significantly. （2）ES-2 cells was shown more invasive cell number when co cultured with supernatants from MA＋thrombin（161.9±11.18,n=6） in comparison of supernatants from MO/MA cultured in medium （47.25±12.45）, which was similar to invasion chemoattracted by original ascites （157.5±14.86,n=4）. Adding hirudin reduced invasive cell numbers （73.5±17.3） significantly. The inhibiting effect of ES-2 invasion presents IL-8 nmAb concentration-dependent. The similar results were also gotten in SKOV3（n=2） and HO-8910（n=2）. （3） Almost all mediators in Inflammation 1 family and Oncogene 3 were activated in MO/MA treated by thrombin （n = 4） ,including NF-Kb p50,p65, c-Fos, c-Rel, c/EBPa, Egr-1, HIF-1, Oct I , Oct Ⅱ. Conclusion： MO/MA stimulated by thrombin enhanced EOC cells invasion ability through IL-8 production. Thrombin maybe involved in EOC cell migration and invasion along peritoneum via educating MO/MA polarization toward tumor associated macrophage like cells.