摘要
目的探讨蛋白酶体抑制剂Z-LLL-CHO(MG132)对人骨肉瘤MG-63细胞诱导凋亡的作用及其对p27Kip1蛋白表达的影响。方法分别以不同浓度的蛋白酶体抑制剂MG132培养p53突变型人骨肉瘤MG-63细胞、正常二倍体成纤维细胞WI-38和p53野生型人骨肉瘤U2OS细胞。通过MTT法检测不同培养时间的细胞增殖活力、琼脂糖凝胶电泳检测细胞凋亡、流式细胞仪定量检测不同时间的细胞凋亡发生率、Westernblot检测p27Kip1表达情况。结果MG132能选择性抑制人骨肉瘤MG-63和U2OS细胞生长,IC50分别为(0.92±0.06)μmol/L及(0.33±0.05)μmol/L,均低于二倍体成纤维WI-38细胞(9.13±0.12)μmol/L。1μmol/LMG132处理MG-63细胞24h后,DNA琼脂糖凝胶电泳可检测到典型的“阶梯状”DNA条带,而WI-38细胞则为基因组DNA。流式细胞仪于1μmol/LMG132作用12h后检测到明显的亚G1峰(凋亡细胞峰),且存在时效关系。Westernblot发现,处理后p27Kip1蛋白表达明显上调。结论蛋白酶体抑制剂MG132在体外可选择性诱导人骨肉瘤MG-63细胞凋亡。p27Kip1蛋白在诱导MG-63细胞凋亡中可能起重要作用。
Objective To investigate the apoptnsis-indueing effect of proteasome inhibitor Z-LLLCHO （MGI32） on human osteosareoma MG-63 cells and study the altered expression of p27^Kipl protein. Methods p53 mutation type hun,an osteosarcoma MG-63 cells, normal diploid fibroblast WI-38 cells and p53 wild type human osteosareoma U2OS cells were cultured with different concentrations of proteasome inhibitor MGI32. Cell viability was determined by MTF assay at different cultured period. Agarose gel electrophoresis was used to detect cell apoptosis and cell apoptotie rate was quantitatively analyzed at different cultured period by flow eytometry in MG-63 and WI-38 cells. Western blot was performed to study the al- tered expression of p27^Kipl protein after treatment. Results MGI32 selectively reduced the viability of human osteosarcoma MG-63 and L2OS cells. The IC50 value was （0.92±0.06） μmol/L and （0.33±0.05） μmol/L, respectively. Moreover, the inhibitory effect was higher on MG-63 or U2OS cells than on diploid fibroblastic WI-38 cells whose IC50 was （9.13±0.12） μmol/L （P〈0.01）. After treatment with 1μmol/L MGI32 for 24 h, the ladder bands characteristic of internncleosomal DNA fragmentation were detected in MG-63 cells but not in WI-38 cells. Apoptotie sub-G1, DNA content was detected by flow cytometry in MG-63 cells incubated with 1μmol/L MGI32 for 12 h and displayed a time-dependent manner. By Western blot, exposure to MGI32 led to an aecumulation of p27^Kipl protein in MG-63 cells. Conclusion Proteasome inhibitor MGI32 had a selective apoptosis-indueing effect on human osteosareoma MG-63 cells in vitro, and altered expres- slon of p27^Kipl protein possibly playing an important role in induction of apoptosis.
出处
《中华骨科杂志》
CAS
CSCD
北大核心
2005年第10期613-617,共5页
Chinese Journal of Orthopaedics
