摘要
[目的]检验恶性骨肿瘤来源的成纤维母细胞是否能够支持破骨细胞分化及其溶骨作用。[方法]酶消化法从肿瘤组织中分离成纤维母细胞并作体外培养。细胞传代后,一部分成纤维母细胞与外周血单核细胞共培养,分别加入M-CSF、骨保护素(OPG)及抗TNFα中和抗体。同时,作者还应用了Transwell系统检验可溶性促溶骨性因子的表达。培养结束后检测抗酒石酸磷酸酶(TRAP)的表达及骨吸收陷窝的形成,并比较各组细胞的骨吸收活性。另一部分成纤维母细胞单独培养,RT-PCR法检测其细胞核因子кB受体活化因子配体(RANKL)和TNFα-mRNA的表达。[结果]在M-CSP存在的条件下,2种细胞共培养组分别于14、21 d观察到TRAP阳性的多核细胞和骨吸收陷窝形成;大剂量OPG彻底阻断TRAP阳性多核细胞及骨吸收陷窝的形成,而大剂量抗TNFα中和抗体对成纤维母细胞支持的破骨细胞形成未显示抑制作用。RT-PCR结果显示恶性骨肿瘤来源的成纤维母细胞能够表达RANKL和TNF-αmRNA。[结论]在M-CSF存在的条件下,恶性骨肿瘤来源的成纤维母细胞通过表达RANKL支持破骨细胞分化及其溶骨作用。
[ Objective] To investigate whether fibroblasts from malignant bone tumor could support osteoclast differentiation and bone resorption. [ Method ] Fibroblasts isolated from 8 fresh tumor samples were cultured in vitro. At passage two, part of these fibroblasts were co-cultured with peripheral blood monocytes. M-CSF, OPG and neutralizing anti-TNF-α antibody was added to the culture respectively. A Transwells system was also applied to observe the secretion of solvable factors in fibroblasts. At day 1, day 14 and day 21, the cultures on coverslips and bone slices were stopped and examined for the formation of tartrate resistant acid phosphatase （TRAP） positive muhmucleated cells （MNCs） and lacuna resorption pits respectively. The number of lacuna resorption pits on bone slices, which was a marker of OC activity, was compared between the groups. Another part of the fibroblasts were cultured alone, and RT-PCR was performed to investigate the expression of RANKL and TNF-α mRNA in the fibroblasts. [Result] TRAP positive MNCs and lacuna pits were observed in the co-culture group in the presence of M-CSF, but not in the absence of M-CSE. It was also the case in the Transwell group, even though the number of lacuna pits was resuced. OPG completely inhibited the formation of TRAP positive MNCs and lacuna pits, while anti-TNF-α had not inhibiting effect on this process. RT-PCR results showed that both RANKL and TNF-α mRNA were positive in all the fibroblast involved. [ Conclusion] In the presence of M-CSF, fibroblasts from malignant bone tumor are capable of supporting osteoclast formation and activity through expression of RANKL.
出处
《中国矫形外科杂志》
CAS
CSCD
北大核心
2006年第5期367-370,共4页
Orthopedic Journal of China
