帕米膦酸二钠抑制骨巨细胞瘤细胞生长和诱导其凋亡

Pamidronate disodium inhibits the growth of bone giant cell tumor and induces its apoptosis

目的:观察第2代二膦酸盐药物——帕米膦酸二钠是否能够抑制骨巨细胞瘤细胞生长以及诱导其凋亡。方法:实验于2004-03/2005-11在北京大学第三医院完成。①标本来源:10例骨巨细胞瘤新鲜手术标本来源于北京大学第三医院骨科和北京积水潭医院骨肿瘤科,均经病理切片确诊。②取肿瘤组织,体外培养原代骨巨细胞瘤细胞。肿瘤细胞贴壁后,分别于培养皿、培养瓶和96孔板内加入帕米膦酸二钠作为用药组,使其终浓度为5,25,50,100,200μmol/L,以磷酸盐缓冲液作为空白对照组,观察骨巨细胞瘤的生长是否受到抑制。③TUNEL法检测骨巨细胞瘤细胞凋亡,以细胞核内出现蓝紫色颗粒为凋亡阳性表现。采用流式细胞仪检测骨巨细胞瘤细胞凋亡率,同时检测半胱氨酸天冬氨酸蛋白酶3蛋白活性的表达情况。结果:①骨巨细胞瘤细胞的培养情况:空白对照组细胞形态正常,胞膜完整;帕米膦酸二钠各浓度组细胞发生皱缩,细胞伪足减少,轮廓不清晰,胞膜不完整,甚至细胞形态消失,发生崩解。②骨巨细胞瘤细胞生长抑制情况:帕米膦酸二钠可抑制骨巨细胞瘤细胞的生长,并且随着作用时间的延长和药物浓度的增加,这种抑制作用增强。③骨巨细胞瘤细胞凋亡TUNEL染色结果:帕米膦酸二钠各浓度组光镜下可见蓝紫色颗粒,空白对照组未见有阳性染色。④流式细胞仪检测骨巨细胞瘤细胞凋亡率结果:帕米膦酸二钠5,50,200μmol/L浓度组作用48h后,细胞凋亡率均明显高于空白对照组[(20.48±6.02)%,(42.39±12.41)%,(54.67±15.38)%,(10.13±3.45)%,P<0.05]。⑤半胱氨酸天冬氨酸蛋白酶3活性检测结果:帕米膦酸二钠5,50,200μmol/L浓度组作用48h后骨巨细胞瘤细胞内的半胱氨酸天冬氨酸蛋白酶3蛋白活性均明显高于空白对照组[(14.93±5.04)%,(25.13±7.60)%,(26.07±7.49)%,(2.73±0.81)%,P<0.05]。结论:帕米膦酸二钠可以抑制骨巨细胞瘤细胞生长,诱导半胱氨酸天冬氨酸蛋白酶3蛋白表达,从而导致骨巨细胞瘤细胞发生凋亡,帕米膦酸二钠对骨巨细胞瘤的作用表现为时间和浓度依赖性。提示应用帕米膦酸二钠治疗和预防骨巨细胞瘤复发具有一定的可行性。

AIM： To observe if the second filial generation of bisphosphonatepamidronate disodium can inhibit the growth and induce the apoptosis of giant cell tumor （GCT） of bone cells. METHODS： The experiment was finished from March 2004 to November 2005 in Third Hospital of Peking University. ①The source of specimen： 10 fresh GCT tumor specimen was obtained from Department of Orthopaedics, Third Hospital, Peking University and Department of Orthopaedic Tumor Surgery of Beijing Jishuitan Hospital. All the specimens were confirmed by pathology. ②The primary GCT cell was gained from the tumor tissue and cultured in vitro, After the tumor cells were adhered, the experiment groups were added with pamidronate disodium into culture dish, culture flask and 96 holes culture plate as medication group at 5, 25, 50, 100, 200 μmol/L, respectively. The blank control group was added with phosphate buffer saline （PBS）. To observe if GCT cell growth was inhibited. ③To detect the apeptosis of GCT cell with TUNEL： It was positive that there was amethyst particle in cell nucleus. The GCT cell apeptosis rate and the activation of caspase-3 were detected with flow cytometer. RESULTS： ①The culture of GCT cell： The cell of blank control group was normal and cell membrane was integrity. The cell of pamidronate disodium group was shrinked, pseudopodia were decreased, the contour was blurred, and the cell membrane was not integrity. The cell form was even disappear and disintegration. ②The inhibition of GCT cell growth： Pamidronate disodium could inhibit the growth of GCT cell. Along with the increase of concentration and lasting of affecting time, it was observed that the inhibition of growth was more obviously. ③TUNEL： The cell was found amethyst particle in cell nucleus of every coneentration of pamidronate disodium group, while the blank control group was not found. ④The apoptosis rate detected by flow cytometer： After treated with pamidronate disodium at 5, 50, 200 μmol/L for 48 hours, the cell apoptosis rate was obviously higher than that in the blank control group [（20.48±6.02）%, （42.39±12.41）%, （54.67±15.38）%, （10.13±3.45）% ,P 〈 0.05]. ⑤The detection findings of caspase-3 activation： After treated with pamidronate disodium at 5, 50, 200 μmol/L for 48 hours, the caspase-3 activation GCT cell was obviously higher than that in the blank control group [（14.93 ±5.04）%, （25.13±7.60）%, （26.07±7.49）%, （2.73±0.81）% ,P 〈 0.05]. CONCLUSION： Pamidronate disodium can inhibit the growth of GCT cell and induce GCT cell apoptosis by stimulating the activation of caspase-3. It was dose-dependent and time-dependent. It is indicated that the pamidronate disodium will be possible used in treatment and prevention of recurrence of GCT.