摘要
目的:明确肿瘤坏死因子超家族成员在骨巨细胞瘤的表达及对其肿瘤细胞的作用,同时观察肿瘤坏死因子相关凋亡诱导配体联合干扰素γ作用于骨巨细胞瘤肿瘤细胞的效果。方法:实验于2005-01/2006-08在解放军总医院骨科研究所完成。①用免疫组织化学染色方法检测肿瘤坏死因子超家族成员核因子κB受体激活因子配基、核因子κB受体激活因子、肿瘤坏死因子相关凋亡诱导配体、死亡受体4、死亡受体5、骨保护素等因子的表达。②骨保护素、肿瘤坏死因子相关凋亡诱导配体、干扰素γ分别以不同的浓度作用于培养的骨巨细胞瘤肿瘤细胞24h;随后用干扰素γ(1000 U/mL)先诱导24h再加入肿瘤坏死因子相关凋亡诱导配体(100,200μg/L)作用于细胞24h,用CCK-8方法来检测细胞存活率。③选取在药物合理浓度范围内有明显抑制作用的用药方式所作用的细胞,用流式细胞仪检测其凋亡率,倒置显微镜下观察细胞药物作用后的形态学改变。结果:①破骨细胞分化相关因子在细胞和组织水平的表达:肿瘤坏死因子相关凋亡诱导配体、骨保护素、核因子κB受体激活因子配基在骨巨细胞瘤所有细胞成分内均有表达,死亡受体4,死亡受体5在多核巨细胞内强阳性表达,在梭型单核细胞表达强度较弱,部分标本内死亡受体4呈阴性表达。核因子κB受体激活因子仅在多核巨细胞内强阳性表达。②CCK-8检测药物作用后细胞抑制作用:骨保护素对肿瘤细胞无抑制作用,肿瘤坏死因子相关凋亡诱导配体仅在较高浓度时对肿瘤细胞有弱的抑制作用,干扰素γ对巨细胞瘤细胞抑制作用有浓度依赖性,当先以干扰素γ(1000 U/mL)诱导24h再加入肿瘤坏死因子相关凋亡诱导配体(100,200μg/L)共同作用24h,细胞存活率明显下降。③凋亡分析:流式细胞检测发现干扰素γ(1000 U/mL)诱导24h再联合不同浓度肿瘤坏死因子相关凋亡诱导配
AIM： To investigate the expression of the tumor necrosis factor （TNF）-Iigand superfamily members in giant call tumor of bone （GCT） tissues and to study its roles in apoptosis of GCT tumor calls in vitro. To demonstrate that tumor necrosis factor-related apoptosls-lnducing ligand （TRAIL） and interferon （IFN） -γ, may act synergistically in GCT tumor calls.
METHODS： The experiment was performed at the Institute of Department of Orthopaedics, General Hospital of Chinese PLA from January 2005 to August 2006. ①The expressions of receptor activator of nuclear factor-κΒ ligand （RANKL）, receptor activator of nuclear factor-κΒ （RANK）, TRAIL, death receptor （DR） 4 and DR5 in GCT tissue were detected by immunohistochemical staining. ②GCT tumor cells were treated by osteoprotegerin （OPG）, TRAIL and IFN-γ, respectively on different concentrations for 24 hours. Cells were pretreatment with IFN -γ, （1 000 u/mL） for 24 hours and further cultured with TRAIL at indicated concentrations （100, 200 μg/L） for 24 hours, and then the survival rate of calls was assayed by CCK-8. ③The tumor calls, which were treated with significant inhibitive effect in a reasonable concentration, were subjected to apoptosis assay by flow cytometry. Invert microscope was applied to observe the morphological changes after drug action.
RESULTS： ①Expression of osteoclast differentiation related factors in cell and tissue levels： RANKL, OPG and TRAIL prOtein expressed In all Cell types of GCT calls. DR4 and DR5 were detected in multinuclear giant cells strongly and spindle-shaped stromal-like tumor calls weakly and partial samples in DR4 showed negative expression, whereas RANK expressed only in multinuclear giant cells. ②Cell inhibition after drug action by CCK-8; OPG had no inhibitive effect on the proliferation of GCT tumor cells. TRAIL could weakly inhibit the GCT tumor calls only on high concentration. IFN-γ caused a dose-dependent effect on inhibition in GCT cells. When pretreatment w
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第2期242-246,T0001,共6页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家高技术研究发展计划(2002AA214081)~~
