【作者】 仲苓芝；

【导师】 李荣贵；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2014， 博士

【摘要】 目的：探讨HO-1在维持人骨肉瘤细胞对三氧化二砷(ATO)抗性中的作用及分子机制。方法：利用实时定量PCR及Western blot检测ATO对人骨肉瘤MG63及U2OS细胞HO-1诱导的表达；利用shRNA介导的RNA干扰技术阻断ATO对MG63及U2OS细胞中HO-1的诱导，研究其对细胞存活、凋亡、活性氧及对ATO敏感性等生物学行为的影响；应用免疫组织化学染色及Western blot等技术探讨ATO诱导HO-1表达的分子机制。结果：首先，发现ATO特异性诱导了人骨肉瘤MG63及U2OS细胞HO-1的高度表达；然后，阻断HO-1的诱导后发现能够显著降低细胞对ATO的抗性，表现为明显降低细胞存活率，表现为细胞的凋亡明显增加，而与凋亡密切相关的细胞内ROS累积增多继而增加细胞对ATO的敏感性；另外，发现ATO上调HO-1的表达是通过增加转录因子Nrf2的入核，促进其在核中的转录活性进而调控下游靶基因HO-1的转录水平而发挥作用的。结论：ATO特异性高幅度诱导了人骨肉瘤细胞HO-1的表达，阻断HO-1的诱导能够显著增加其对化疗药物ATO的敏感性。ATO诱导HO-1表达的分子机制是通过增加转录因子在细胞核中的转录活性，进而调控下游靶蛋白HO-1的表达来发挥作用的。HO-1在维持人骨肉瘤细胞对化疗药物ATO抗性中具有重要的作用，表现为通过清除肿瘤细胞内活性氧的累积继而抑制细胞的凋亡，从而促进细胞对ATO的耐药性。研究结果为研究骨肉瘤的生物学行为及分子机制提供了新的思路，为靶向HO-1的基因治疗配合药物化疗提供了理论依据。更多还原

【Abstract】 1Highly induction of HO-1by ATO occurs specifically in human osteosarcomaMG63and U2OS cellsArsenic trioxide (ATO) has been successfully used to treat leukemia and some solidmalignant tumors. But the effect to mesenchymal-derived human osteosarcoma cells andrelevant mechanism are unclear. From the toxic effects of arsenic trioxide on normalcells, oxidative damage plays a major role, so we first test changes of antioxidantgenes in human osteosarcoma MG63and U2OS cells under the induction of arsenictrioxide. Objective: To detect changes of antioxidant genes in human osteosarcomaMG63and U2OS cells which were treated with chemotherapy drugs arsenic trioxide.Methods: We used real-time PCR and Western blot methods to detect the mRNA andprotein expression of several kinds of antioxidant related genes after treatment witharsenic trioxide for24h. Results: HO-1mRNA was increased to more than80and120folds in human osteosarcoma MG63and U2OS cells respectively (P<0.01), whileother detected antioxidant genes changed little (P>0.05). HO-1protein was increasedto more than6and8folds in human osteosarcoma MG63and U2OS cellscorrespondingly (P<0.01). HO-1mRNA was increased to less than8folds and HO-1protein was increased to less than2folds in HUVECs after exposure to ATO.Conclusions: HO-1was highly induced in human osteosarcoma cells upon treatmentof ATO specifically.2MG63and U2OS cells were transfected with shRNA-based vectors and stablesilenced cellclones were establishedATO could induce HO-1of human osteosarcoma cells highly expressspecifically. Since HO-1could remove reactive oxygen species (ROS) directly, andROS played an important role in apoptosis, we speculated that HO-1was contactedwith the resistance of osteosarcoma cells of arsenic trioxide. Objective: To clarify therole of gene HO-1in chemotherapy of osteosarcoma cells for arsenic trioxide, wedesigned and synthesized short hairpin based plasmid targeting gene of HO-1. Methods: We transfected the shRNA-mediated plasmid and scramble plasmid to MG63andU2OS cells by lipofectin transfection techniques. We got several single cell clonesthrough the resistance of G418and expression of red fluorescent protein. Results andconclusions: The stable silenced cell clones were established by shRNA-mediatedinterference.3HO-1silencing enhanced ATO-mediated cell death in MG63and U2OSosteosarcoma cellsObjective: To investigate the role of HO-1in susceptibility of humanosteosarcoma cells to arsenic trioxide. To detect if there is any difference inHO-1-shRNA, scramble and parent cell groups, in order to study the pathogenesis ofosteosarcoma with multidrug-resistance and look for effective therapeutic measures.Methods: We analyzed the expression of gene HO-1in MG63and U2OS cells byreal-time PCR and Western blot after transfection. We used CellTiter-Glo assay todetect cell activity and IC50values of arsenic trioxide, and used AnnexinV-FITC/7-AAD and TUNEL assay to detect apoptotic cells, and determinedintracellular accumulation of reactive oxygen species with the ROS detection kit.Results: HO-1was silenced mainly in human osteosarcoma cells in spite of treatmentupon arsenic trioxide. Compared with parent and scramble groups, the apoptosis rateand intracellular accumulation of reactive oxygen species in HO-1silenced cellsincreased significantly in a dose-dependent and time-response effect (P<0.05). TheIC50values of HO-1-shRNA cells to arsenic trioxide were lower than parent andscramble cells (P<0.05). Conclusions: The resistance to arsenic trioxide of humanosteosarcoma MG63and U2OS cells was closely associated with the expression ofHO-1. Silencing HO-1increased the sensitivity of human osteosarcoma MG63cells tochemotherapeutic drug arsenic trioxide, by decreasing the cell viability, increasing the cellapoptosis rate, and enhancing intracellular accumulation of ROS. The results suggested thattargeting HO-1combined with chemotherapy drugs ATO supply a new way for the clinicaltreatment of osteosarcoma.4The molecular mechanisms involved in which HO-1was highly induced by ATOObjective: To investigate the molecular mechanisms of HO-1high expressioninduced by arsenic trioxide in human osteosarcoma cells. Methods: We used theinhibitor of mRNA polymeraseⅡ, Actinomycin-D (Act-D), and histone deacetylaseinhibitors to interfere with induction of HO-1by arsenic trioxide, in order to exclude interference of the stability of mRNA and the impact of histone acetylation. Weanalyzed HO-1expression by qRT-PCR and western blot. We extracted the protein oftranscription factor Nrf2with nucleoprotein extraction kit and used western blot andimmunocytochemistry to detect the expression of Nrf2in nucleus and cytoplasm.Results: Gene transcription inhibitors (Act-D) inhibited the induction of HO-1byarsenic trioxide significantly (P<0.01), suggesting that the increasing expression ofHO-1has nothing to do with stability of the mRNA; While TSA, sodium butyrate orother histone deacetylase inhibitors couldn’t change the HO-1expression apparently(P>0.05), suggesting the regulation in transcription has no obvious correlation withhistone acetylation. Along with the expression of HO-1increased, transcription factorNrf2targeting to antioxidant genes including HO-1expressed more in cell nucleus(P<0.01), and changed scarcely overall (P>0.05). Conclusions: Arsenic trioxideplayed a role in regulating the level of transcription on HO-1, and has nothing to dowith histone acetylation. The involved molecular mechanism was by increasing theexpression of transcription factor Nrf2in the nucleus and enhancing transcriptionlevel of target gene downstream exactly as HO-1.更多还原