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TRAIL及联合阿霉素治疗骨肉瘤的动物实验研究

Treatment of osteosarcoma with TNF-related apoptosis-inducing ligand or in combination with doxorubicin in animal experiment

摘要:

目的 探讨肿瘤坏死因子相关凋亡诱导配体(TRAIL)以及TRAIL联合阿霉素(ADM)对裸鼠移植性骨肉瘤的治疗作用及机制.方法 取对数生长的MG-63骨肉瘤细胞,以5×10~6/ml浓度的细胞悬液0.2 ml接种于裸鼠下肢外侧皮下,建立裸鼠肿瘤模型随机分组.各以1、2 μg TRAIL,15 μg ADM,1μ,g TRAIL+15μg ADM和100 μl生理盐水腹腔注射,观察肿瘤生长速度,处死裸鼠取血清,应用血清碱性磷酸酶(ALP)试剂盒检测血清ALP的活性,取下瘤体应用TUNEL技术检测瘤体中细胞凋亡情况,应用免疫组化法检测Bax基因的表达,应用RT-PCR技术分析TRAIL受体表达的情况.结果 肿瘤生长曲线显示:腹腔注射TRAIL蛋白组与注射生理盐水组相比,裸鼠皮下移植瘤生长更慢,且2 μg TRAIL组的抑制作用强于1μg TRAIL组;TRAIL与ADM联用较两种药物单用具备更强的抑瘤作用.各实验组间ALP活性差异统计学意义.细胞凋亡的TUNEL检测显示TRAIL与ADM联用组较两种药物单用组有更多的凋亡细胞出现.免疫组化检测Bax基因的表达显示TRAIL与ADM联用出现Bax蛋白表达量上调.RT-PCR检测显示TRAIL与ADM联用出现TRAIL-R2的mRNA表达上调.结论 在活体水平TRAIL具有促进骨肉瘤细胞凋亡、抑制肿瘤生长的作用,且这种作用一定范围内随药物的剂量提高而增强.TRAIL与ADM联用具备协同效应,其机制与TRAIL-R2的mRNA和Bax基因的表达上调有关.

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abstracts:

Objective To examine the effect of TNF-related apoptosis-inducing ligand(TRAIL)and in combination with doxorubidcin (ADM) to xenografted tumors in nude mice and to explore its potential mechanism.Methods MG-63 cells(5×10~6/ml) were suspended in 0.2 ml RPMI-1640 and inoculated subcutaneously into the lower limb of nude mice.Treatment groups were given TRAIL of different concentration or combination of TRAIL and ADM intraperitoneally.Normal saline was administrated in the control group.Auti-tumor effects were estimated by tumor volumes.Serum alkaline phosphatase(ALP)was detected by ALP kits.Induction of apoptosis in xenografted tumors was confirmed by TUNEL(TdT-mediated dUTP nick end labeling)assay.Expression of Bax was detected by immunohistochemical assay.Expression of TRAIL receptors was detected by RT-PCR assay.Results Growth curve of tumors indicated that tumors carried by TRAIL-treated mice grew more slowly than that with normal saline and 2μg TRAIL was more effective,Also tumors treated with combination of TRAIL and ADM grew more slowly than any other group.ALP activities of each group were moderately different but significance was not reached.TUNEL showed that there were more apoptotic cells in the combination group than any other group.Immunohistochemical assay showed that expression of Bax was up-regulated in the combination group.RT-PCR showed that expression of TRAIL-R2 mRNA was up-regulated in the combination group.Conclusion TRAIL can induce an effective apoptosis of osteosarcoma cells in vivo in a dose-dependent fashion.ADM can enhance the effect of TRAIL-mediated apoptosis.And up-regulations of Bax and TRAIL-R2 may be the involved mechanism.

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作者: 吴刚 [1] 喻爱喜 [1] 祝少博 [1] 漆白文 [1]
期刊: 《中华医学杂志》2009年89卷37期 2644-2648页 MEDLINEISTICPKUCSCD
分类号: R9
栏目名称: 基础研究
DOI: 10.3760/cma.j.issn.0376-2491.2009.37.013
发布时间: 2009-11-30
基金项目:
湖北省卫生厅青年科技人才项目 教育部高等学校博士学科点专项科研基金
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