二膦酸盐对骨巨细胞瘤细胞的生长抑制和诱导凋亡作用

The experimental study of bisphosphonates inducing the expression of caspase-3 in the cells of bone giant cell tumor

目的观察二膦酸盐对骨巨细胞瘤细胞生长的影响。方法取10例手术切除的骨巨细胞瘤新鲜组织进行原代培养，待瘤细胞贴壁生长后分别给予5、25、50、100、200μmol／L阿仑膦酸钠、帕米膦酸二钠。作用一定时间后，用倒置相差显微镜观察细胞形态，MTT法检测细胞活性，末端酶标记法凋亡染色观察细胞凋亡情况，流式细胞术检测凋亡率和caspase-3表达。结果（1）二膦酸盐作用后，细胞正常形态消失，甚至发生崩解。（2）药物作用24h后，阿仑膦酸钠可以抑制细胞活性的2．79％±0．92％-31．17％±8．05％，帕米膦酸二钠为5．87％±1．94％-39．68％±9．26％；48h后，阿仑膦酸钠可以抑制细胞活性的8．68％±2．87％—46．88％±11．24％，帕米膦酸二钠为10．49％±3．26％～60．43％±13．64％；72h后，阿仑膦酸钠可以抑制细胞活性的11．13％±3．60％-49．94％±11．67％，帕米膦酸二钠为15．57％±5．86％～63．97％±16．42％。（3）药物作用48h后，阿仑膦酸钠和帕米膦酸二钠5μmol／L组的细胞凋亡率分别为16．25％±5．62％、20．48％±6．02％；50μmol／L组分别为37．20％±12．01％、42．39％＋12．41％；200μmol／L组分别为47．53％＋13．92％、54．67％＋15．38％。（4）帕米膦酸二钠作用48h后，5、50、200μmol／L组瘤细胞的caspase-3活性分别为14．93％±5．04％、25．13％±7．60％、26．07％＋7．49％，引起的细胞凋亡表现为时间和浓度依赖性。结论二膦酸盐可能成为治疗和预防骨巨细胞瘤复发的一个治疗方法。

Objective To evaluate the inhabitation of cell proliferation and apoptosis of giant cell tumor induced by bisphosphonates in vitro. Methods Ten tissue samples were harvested at the time of surgery from patients undergoing tumor resection. After the majority of tumor cells adhered to the culture flasks surface, Alendronate and pamidronate was used as test storage media at 5, 20, 50, 100, 200μM respectively. Cells viability was detected by MTT Assay after the tumor cells were cured with different concentration at different time periods. To detect tumor cells apoptosis with in situ TUNEL assay and flow cytometry. The active caspase-3 was also detected. Results After exposure to bisphosphonates, the cells exhibited the characteristic features of cell shrinkage, rounding and partial detachment, and demonstrated the lobulated appearance of apoptotic cells. It was much more prominent while the treating time prolonged or the concentration increased. Alendronate （5-200 μmol/L） treatment for 24 h, resulted in 2.79%±＋0.92% to 31.17%± 8.05% decrease in cell viability, 8.68%±2.87% to 46.88%±11.24% for 48 h, and 11.13%±3.60% to 49.94%±11.67% for 72 h, respectively. Pamidronate （5-200μmol/L） treatment for 24 h, resulted in 5.87%±1.94% to 39.68%±9.26% decrease in cell viability, 10.49%±3.26% to 60.43%±13.64% for 48 h, and 15.57%±5.86% to 63.97%±16.42% for 72 h, respectively. A significant dose-dependent and time-dependent decrease in the number of viable cells was observed in the GCT cells. After bisphosphonate treatment for 48 hours, the mean cell population in apoptosis of alendronate and pamidronate was 16.25%±5.62% ,20.48%±6.02% at concentration 5μmol/L, 37.20%±12.01%, 42.39%±12.41% at concentration 50μmol/L, and 47.53%±13.92%, 54.67%±15.38% at concentration 200μmol/L respectively. In bisphosphonates-treated GCT cells, caspase-3 activation was observed. After treated by pamidronate for 48 h, caspase-3 expression was 14.93%±5.04%, 25.13%±7.60%, 26.07%±7.49% at 5, 50, 200μmol/L respectively. It was dose and time dependent response. Conclusion Bisphosphonates would be a new adjuvant treatment and prevention of recurrence besides the operation of giant cell tumor of bone.