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目的构建复制缺陷型腺病毒N-烷基嘧啶DNA糖基化酶(MPG)表达载体,观察人骨肉瘤细胞HOS过表达MPG及其对DNA损伤药物甲基甲磺酸酯(MMS)、N-甲基-N1-硝基-N-亚硝基鸟嘌呤(MNNG)和替莫唑胺(TMZ)敏感性的影响。方法应用流式细胞术、蛋白印迹和HEX标记寡核苷酸方法确定MPG腺病毒表达载体Ad5HA-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、硫氰酸盐B法和[3H]胸腺嘧啶掺入法检测经MMS(1、2μmol/L)、MNNG(25、50μmol/L)和TMZ(1、2mmol/L)处理过的转染细胞存活情况;PE-Annexinv/7-AAD流式细胞术检测细胞凋亡。结果10个感染增殖指数(MOI)的Ad5HA-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,其半数抑制浓度(IC50)值分别下降了6·0、4·5和2·5倍。结论Ad5HA-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 To construct replication-defective adenovirus N-alkyl pyrimidine DNA glycosylase (MPG) expression vector and to observe HOS overexpression of MPG in human osteosarcoma cells and its effect on DNA damage drug methyl mesylate (MMS), N-A Effects of Sensitivity of Sigma-N1-Nitro-N-Nitrosoguanine (MNNG) and Temozolomide (TMZ). Methods Flow cytometry, Western blot and HEX-labeled oligonucleotides were used to determine the efficiency of MPG adenovirus expression vector Ad5HA-MPG in HOS of human osteosarcoma cells, MPG protein expression and MPG enzyme activity; MTS, thiocyanate The survival rate of transfected cells treated with MMS (1, 2 μmol/L), MNNG (25, 50 μmol/L) and TMZ (1, 2 mmol/L) was detected by the method of salt B and [3H] thymidine incorporation; - Annexinv/7-AAD flow cytometry was used to detect apoptosis. Results More than 90% of the HOS infected with Ad5HA-MPG infected with the proliferation index (MOI) could be infected. The infected cells expressed MPG highly and had MPG enzyme activity. Overexpression of HOS in MPG significantly increased the sensitivity of DNA-damaging chemotherapeutic drugs MMS, MNNG and TMZ, and their IC50 values decreased by 6.0, 4-5, and 2.5 times, respectively. Conclusion Overexpression of transient MPG in Ad5HA-MPG may be a potential therapeutic method to increase the sensitivity of osteosarcoma cells to DNA-damaging chemotherapeutic drugs.