HMME介导的光动力疗法对骨肉瘤细胞的杀伤效应及机制研究  

Study on the killing effect and mechanism of hematoporphyrin monomethyl ether mediated photodynamic therapy on osteosarcoma cells

作　　者：曾辉[1] 孙梦熊[1] 王卓莹[1] 廖宇昕[1] 付东[1] 吴非[1] 华莹奇[1] 蔡郑东[1] 

机构地区：[1]同济大学附属第十人民医院骨科,上海200072

出　　处：《中国骨与关节杂志》2012年第5期494-500,共7页Chinese Journal of Bone and Joint

基　　金：上海市科学技术委员会基金资助项目(11JC1410101)

摘　　要：目的探讨血卟啉单甲醚介导的光动力疗法(HMME-PDT)对骨肉瘤LM-8细胞增殖与凋亡的影响及其机制。方法将LM-8细胞与不同浓度的HMME(10、20、30μg/m1)共同孵育,4h后,给予不同能量密度的激光(3、6、9J/cm^2)照射,同时设空白组(无光敏剂也无光照)、暗毒组(无光照但加入光敏剂)、激光组(不加光敏剂但给予光照)。MTT法检测细胞存活率,采用AnnexinV-FITC/PI染色的流式细胞分析法检测LM-8细胞凋亡和周期情况,Hoechst33342染色法观察细胞凋亡,实时定量PCR(QRT-PCR)分别检测Caspase-3、Bcl-x、p53、RelA mRNA表达水平,免疫印迹(Western blotting)分别检测Caspase-3、Bcl-x、p53、RelA蛋白的相对表达量。结果 HMME-PDT能显著抑制骨肉瘤LM-8细胞增殖,且杀伤效应随着HMME浓度及能量密度的增高而增强。然而单独光照或单独给予光敏剂孵育,对骨肉瘤LM-8细胞均无杀伤作用。流式细胞术检测发现凋亡率显著增高,10μg/ml HMME组为(12.3±2.82)%,20μg/ml HMME组为(20.67±5.58)%,30μg/ml HMME组为(42.53±4.64)%,凋亡率与空白组比较,结果分别为(t=-2.889,P=0.045)、(t=-4.418,P=0.014)、(t=-5.780,P=0.04)。周期结果显示:G_0/G_1期细胞明显增高、S期细胞明显降低,30μg/ml组G_0/G_1期为(50.09±3.15)%、S期为(28.17±1.10)%,与空白对照组比较,结果分别为(t=-6.081,P=0.004)、(t=5.852,P=0.004)。HMME-PDT处理LM-8细胞后经Hoechst33342染色后呈现出典型的凋亡改变(如:核固缩、细胞变网等)。QRT-PCR和Western bloting结果显示:HMME-PDT可显著上调Caspase-3 mRNA和蛋白相对表达量且表达量随着HMME浓度增高而增高。而HMME-PDT可明显下调Bcl-x、p53、RelA mRNA和蛋白相对表达量且表达量随着HMME浓度增高而降低。结论在体外HMME-PDT对骨肉瘤LM-8细胞有显著的杀伤效应,其杀伤效应与Caspase-3、Bcl-x、p53和RelA密切相关。Objective To investigate the effect and mechanism of hematoporphyrin monomethyl ether mediated photodynamic therapy （HMME-PDT） on the proliferation and apoptosis of osteosarcoma cell line LM-8. Methods LM-8 cells were cultured with different concentrations of HMME （10, 20, 30pg/ml） for 4 hours, and then illuminated by laser at various energy densities （3, 6, 9J/cm：）. And meanwhile, the blank group （without photosensitizer or irradiation）, photosensitizer group （with photosensitizer but without irradiation） and laser group （with irradiation but without photosensitizer） were selected as control groups. The cell viability was measured by 3-（4, 5-Dimetbylthiazol- 2-yl）-2, 5-diphenyltetrazolium bromide, a yellow tetrazole （MTT） assay. The apoptosis and cycle of LM-8 cells were detected by flow cytometry method with Annexin V-FITC/PI staining. The apoptosis of LM-8 cells was further observed by Hoechst33342 staining. The expression levels of Caspase-3, Bcl-x, p53 and RelA mRNA were detected by real-time quantitative reverse transcription-polymerase chain reaction （QRT-PCR）. Western blot test was used to examine the relative expressions of Caspase-3, Bcl-x, p53 and RelA proteins. Results HMME-PDT markedlyinhibited the proliferation of osteosarcoma cell line LM-8, and the killing effect improved with the increase of HMME concentration and energy intensity. However, with laser irradiation or photosensitizer alone, HMME-PDT showed no killing effect on osteosarcoma cell line LM-8. The flow cytometry method revealed the apoptosis rates in the groups significantly increased, which were （12.3±2.82）%, （20.67±5.58）% and （42.53±4.64）% respectively in the groups treated with 10, 20 and 30gg/ml HMME. Comparing with the blank group, the results were （t=-2.889, P=0.045）, （t=-4.418, P=0.014）, and （t=-5.780, P=0.04） respectively. The results of cell cycle analysis showed a significant increase of the number of cells in G0/G1 phase and an obvious decrease of the proportion

关 键 词：光动力疗法 骨肉瘤 HMME 凋亡 基因 

分 类 号：R738.1[医药卫生—肿瘤] R730.57[医药卫生—临床医学]