双膦酸盐诱导骨巨细胞瘤细胞凋亡的实验研究

Experimental study of bisphosphonate(zoledronate) inducing apoptosis of giant cell tumor of bone stromal cell and multinucleated giant cells

目的探讨双膦酸盐(唑来膦酸)对骨巨细胞瘤多核巨细胞和基质细胞的诱导凋亡作用。方法对我院收治的7例唑来膦酸治疗前后患者的肿瘤组织切片进行透射电镜观察和TUNEL凋亡染色及图像分析。另10例未予唑来膦酸治疗的患者,取术中切除的骨巨细胞瘤新鲜组织进行细胞原代培养,分别给予5、30、150μmol/L唑来膦酸诱导肿瘤细胞凋亡48h,倒置相差显微镜观察细胞形态,Annexin-V FITC凋亡染色及流式细胞仪定量分析。结果唑来膦酸治疗后的肿瘤组织切片透射电镜观察到多核巨细胞和基质细胞呈明显的凋亡表现;TUNEL染色呈强阳性;基质细胞凋亡率治疗前为1.31%,治疗后33.42%(P=0.018);多核巨细胞凋亡率治疗前为8.41%,治疗后56.83%(P=0.018)。在原代培养的骨巨细胞瘤基质细胞中予不同浓度的唑来膦酸诱导凋亡48h后,细胞数量减少,部分细胞形态有显著变化;Annexin-V FITC凋亡染色及流式细胞仪定量分析结果显示,对照组以及5、30、150μmol/L唑来膦酸诱导组的凋亡率分别为(10.67±2.15)%、(20.47±3.77)%、(44.21±8.34)%和(56.96±9.68)%,各诱导组显著高于对照组(P<0.05),且凋亡率呈剂量依赖性(r=0.775,P=0.000)。结论双膦酸盐能够诱导骨巨细胞瘤基质细胞和多核巨细胞的凋亡,且骨巨细胞瘤基质细胞的凋亡率呈剂量依赖性,因此,双膦酸盐可以作为骨巨细胞瘤辅助治疗的方法之一。

Objective To investigate the feasibility of bisphosphonates（zoledronate） inducing apoptosis of giant cell tumor of bone（GCTB）stromal cell and multinucleated giant cells. MethodsIn this study,the feasibility of zoledronate inducing apoptosis of GCTB stromal cell and multinucleated giant cells of GCTB was explored with cell culture,microscopy,electron microscopy and TUNEL apoptosis staining and image analysis,flow cytometry quantitative analysis of Annexin-V apoptosis index.GCTB specimens were collected at the time of biopsy and during definitive surgery.TUNEL assay was used to evaluate apoptotic DNA fragmentation in cells.In addition,ten GCTB primary cultures from these patients were treated with zoledronate for 48 hours at different doses（5μmol/L,30μmol/L,and 150μmol/L） and subjected to apoptosis assay by flow cytometry following fluorescent Annexin-V labelling.Transmission electron microscopy was used to identify ultrastructural changes,indicative of apoptosis in the cytoplasm and the nucleus of the giant cells. ResultsZoledronate significantly induced apoptosis in both osetoclast-like giant cells and stromal tumor cells.Significant changes were observed in tumour samples under the transmission electron microscopy（TEM）.The strong positive TUNEL-apoptosis staining were observed in the tumor samples after zoledronate administration.The apoptosis rate of stromal tumor cells increase significantly from 1.31% to 33.42% after zoledronate administration when the apoptosis rate of osetoclast-like giant cells increase significantly from 8.41% to 56.83% after zoledronate administration;In zoledronate-induced group,GCTB stoma cell line induced by zoledronate for 48 hours decreased in quantity,highlighted the changes in morphology.The apoptosis rates of GCTB in the apoptosis assay by flow cytometry following fluorescent Annexin-V labelling increased from 10.67% in control group to 20.47%,44.21%,56.96% at 5μmol/L,30μmol/L,and 150μmol/L zoledronate induction groups in vitro.Zoledronate caused substantial apoptosis of stromal tumor cells in dose-dependant manner. ConclusionBisphosphonates induces apoptosis of GCTB stromal cell and multinucleated giant cells in dose-dependant manner.Bisphosphonates may be considered as potential adjuvants in the treatment of GCTB.