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目的本研究首先观察N-烷基嘧啶DNA糖基化酶(MPG)在人体骨肉瘤中的表达特点及其与患者预后的关系,进而构建复制缺陷型腺病毒MPG表达载体,探讨人骨肉瘤细胞HOS过表达MPG及其对DNA损伤药物MMS,MNNG和TMZ敏感性的影响。方法应用免疫组化方法检测60例骨肉瘤组织和3株骨肉瘤细胞中MPG的表达。按染色强度将其分为低表达组和高表达组,统计分析它们与临床病理及患者预后的关系。应用流式细胞术、蛋白印迹和HEX标记寡核苷酸方法确定MPG腺病毒表达载体Ad5 HA-MPG在人体骨肉瘤细胞HOS中的感染效率,MPG蛋白表达和MPG酶活性;MTS、SRB和[3H]胸腺嘧啶掺入法检测细胞存活;PE-Annexinv/7-AAD流式细胞术检测细胞凋亡。结果3株骨肉瘤细胞MPG均呈弱阳性表达。60例骨肉瘤中MPG 40例(65%)为高表达,MPG表达和WHO分型和患者预后显著相关。10 MO I Ad5 HA-MPG可使90%以上HOS感染,感染细胞高表达MPG并具有MPG酶活性。MPG过表达HOS显著地提高DNA损伤性化疗药物MMS、MNNG和TMZ的敏感性,其IC50值分别下降了6.0、4.5和2.5倍。结论Ad5 HA-MPG瞬时MPG过表达,可能是一种提高骨肉瘤细胞对DNA损伤性化疗药物敏感性的潜在治疗方法。
Objective This study first observed the expression of N-alkyl pyrimidine DNA glycosylase (MPG) in human osteosarcoma and its relationship with the prognosis of patients, and then construct replication-deficient adenovirus MPG expression vector to explore human osteosarcoma cells HOS Over-expression of MPG and its effect on the DNA damage agents MMS, MNNG and TMZ sensitivity. Methods Immunohistochemistry was used to detect the expression of MPG in 60 cases of osteosarcoma and 3 cases of osteosarcoma. According to their staining intensity, they were divided into low expression group and high expression group, and their relationship with clinical pathology and prognosis were statistically analyzed. The infection efficiency, MPG protein and MPG activity of MPG adenovirus expression vector Ad5 HA-MPG in HOS of human osteosarcoma cells were determined by flow cytometry, Western blot and HEX-labeled oligonucleotide method. MTS, SRB and [ 3H] thymidine incorporation assay; cell apoptosis was detected by PE-Annexinv / 7-AAD flow cytometry. Results Three osteosarcoma cells showed weak positive expression of MPG. In 60 cases of osteosarcoma MPG 40 cases (65%) were high expression, MPG expression and WHO classification and prognosis of patients was significantly related. 10 MO I Ad5 HA-MPG infected more than 90% of HOS infected cells with high MPG expression and MPG enzyme activity. Over-expression of HOS by MPG significantly increased the sensitivity of DNA-damaging chemotherapeutic agents, such as MMS, MNNG and TMZ, with IC50 values of 6.0, 4.5 and 2.5 fold lower, respectively. Conclusion Transient overexpression of Ad5 HA-MPG may be a potential therapeutic approach to improve the sensitivity of osteosarcoma cells to DNA-damaging chemotherapeutic drugs.