加味三棱丸对子宫内膜异位症雌激素生成及内膜侵袭能力的影响

Inhibitory effect on estrogen production and its influence on invasive ability of human endometrial cells of endometriosis by medicated serum of SLW

目的:明确加味三棱丸(SLW)抗子宫内膜异位症(内异症)雌二醇生成的作用,并探讨其在SLW抑制内膜细胞侵袭过程中所起的关键作用。方法:培养子宫肌瘤和内异症患者在位子宫内膜细胞,以子宫肌瘤为对照,内异症在位内膜细胞经SLW大鼠血清高、中、低剂量处理后,分别在不同的时间点采用电化学发光免疫法检测雌二醇(E2)的分泌水平;明胶酶谱法分析内膜细胞分泌的基质金属蛋白酶2,9(MMP-2,9)活性,细胞免疫荧光法观察组织型金属蛋白酶抑制剂1,2(TIMP-1,2)蛋白的表达;通过时效关系的考察,明确SLW抑制内膜细胞侵袭能力的最佳时间后,重新分为6组:子宫肌瘤组、内异症组、内异症+SLW中剂量组、内异症+SLW中剂量+雌二醇组、内异症+阿那曲唑血清组和内异症+雌二醇组,分别在最佳时间点检测各组细胞分泌的MMP-2,9活性及TIMP-1,2蛋白表达水平。结果:SLW高、中剂量可明显降低内异症在位内膜细胞分泌E2的水平,并呈时间和浓度依赖性;内异症组在各检测时间内,分泌的MMP-2,9活性明显高于子宫肌瘤内膜组(P<0.01);经SLW血清中剂量处理48 h后,与空白对照组比较,内异症在位内膜细胞分泌的MMP-2,9活性明显降低(P<0.01),而TIMP-1,2蛋白的表达明显升高(P<0.01);当SLW中剂量与雌二醇2.70 ng.L-1联合处理48 h后,内异症在位内膜细胞被SLW降低的侵袭能力可被反向添加的雌二醇部分恢复;其中,MMP-2活性、TIMP-1,2蛋白表达与空白组比较,已无显著性差异;而单独进行雌二醇处理,可进一步增加内异症内膜细胞的侵袭能力,特异性芳香化酶抑制剂阿那曲唑与雌二醇的作用恰好相反。结论:SLW可抑制内异症在位内膜细胞的侵袭能力,其作用与SLW降低内膜细胞雌二醇的分泌密切相关。

Objective： To study the inhibitory effect of medicated serum of SLW on estrogen production and to approach on the key mechanism of SLW in inhibiting the invasion ability of endometrial cells of endometriosis.Method： First,the model of eutopic primary cultured endometrial cells of endometriosis and hysteromyoma in vitro was successfully established.Taking that of endometrial cells of hysteromyoma as control,the secretion level of E2 of endometrial cells in the culture media supernatant at different time point with the treatment of high,middle and low dose of SLW serum was detected by electrochemiluminescence immunoassay,the activities of matrix metalloproteinase-2,9（MMP-2,9） were detected by gelatinase zymography assay,and the expression of tissue inhibitor of metalloproteinase-1,2（TIMP-1,2） protein was observed by immunofluorescence.After the optimal time for SLW to inhibit invasion ability of endometrial cells was identified based on time-effect relationship,another endometrial cells were divided into six groups： hysteromyoma endometrium group,eutopic endometrium of endometriosis group,eutopic endometrium of endometriosis ＋middle dose of SLW serum group,eutopic endometrium of endometriosis ＋ middle dose of SLW serum ＋ E2 group,eutopic endometrium of endometriosis ＋ anastrozole serum group,and eutopic endometrium of endometriosis ＋ E2 group.The activities of MMP-2,9 and the expression of TIMP-1,2 protein were detected according to the optimal time point.Result： The secretion level of E2 of eutopic endometrium in endometriosis could be decreased by SLW,which showed the dependence of time and concentration.The result of gelatinase zymography assay and immunofluorescence respectively showed that along with the time the activities of MMP-2,9 of eutopic endometrial cells of endometriosis were significantly higher than those of hysteromyoma at the same time point（P0.01）.After 48 hours,with the treatment of middle dose of SLW serum,the activities of MMP-2,9 of eutopic endometrial cells of endometriosis could be decreased（P0.01） while the expression of TIMP-1,2 protein could be increased obviously（P0.01）.The malignant invasion ability improved by SLW of eutopic endometrial cells of endometriosis was partly recruited by add-back E2 treatment.There was no significant difference in the activity of MMP-2 and the expression of TIMP-1,2 protein between eutopic endometrium of endometriosis ＋ middle dose of SLW serum ＋ E2 group and untreated group.The behavior of invasion of endometrial cells of endometriosis could be deteriorated treated by E2 as contrast to anastrozole,a specific aromatase inhibitor.Conclusion： SLW could decrease the secretion of E2 so as to inhibit the invasion of the eutopic endometrial cells of endometriosis.