氢醌依赖NF-κB信号转导途径对骨髓间充质干细胞MDR1基因和P-糖蛋白表达的影响

Over expression of P-gp encoded by MDR1 is associated with acting to pump the xenobiotics out of cells and protects cells from damage. Nuclear factor-kappa B (NF-κB) is a crucial transcriptional factor that plays a major role in regulating transcription of MDR1. Bone mesenchymal stem cells (BMSCs) modified geneticly can make the significant efficacy in many human disorders in various ways. We previously reported that people exposed to benzene with MDR1 3435TT genotype had the decreased white blood cell counts. The expression of MDR1 and P-gp on bone monocytes of C57BL/6 mice on gavage with benzene was down regulated. Based on these building blocks, we get ready to carry out the following three methods: 1) To detect the expression of MDR1 and P-gp on bone mesenchymal stem cells of rabbits after apoptosis induced by HQ treatment in vitro; 2) To analyze the regulation on MDR1 transcription and P-gp expression by phosphorylation, translocation and p65, p50 expression of NF-κB; 3) To investigate the influence of MDR1 over expression or gene silence on apoptosis induction of BMSCs. Our purpose is to illustrate the possible mechanism that whether MDR1 and P-gp get to involve in the benzene hematology toxicity with NF-κB signal transduction pathway. If the hypothesis is confirmed, one new strategy to control the chronic benzene poisoning will become possible.

MDR1基因编码的P-gp使细胞泵出外源性物质，保护细胞免受损害，NF-κB是调控MDR1转录的重要信号途径，骨髓间充质干细胞（BMSCs）经遗传修饰后可以不同形式治疗疾病。我们在前期研究中发现，携带MDR1 3435 TT基因型的苯作业人员WBC下降，苯染毒小鼠骨髓单个核细胞MDR1基因、P-gp表达下调，那么苯是否影响BMSCs MDR1基因和P-gp表达，并通过何种信号途径进行调节？本研究拟采用：1）HQ体外染毒兔BMSCs，观察BMSCs凋亡与BMSCs的MDR1基因、P-gp表达的相关性；2）NF-κB亚基p65的表达、磷酸化和转位对MDR1基因转录、P-gp表达的调控；3）MDR1基因过表达或表达沉默，对苯所致BMSCs凋亡的影响，初步阐明MDR1基因和P-gp是否依赖NF-κB信号途径参与苯的血液毒性，寻找慢性苯中毒治疗的新靶点。

MDR1 基因编码的P-gp 使细胞泵出外源性物质，保护细胞免受损害，NF-κB 是调控MDR1 转录的重要信号途径，但在苯血液毒性中的作用研究甚少，本研究通过苯的活性代谢产物HQ 体外染毒兔BMSCs，观察骨髓间充质干细胞（BMSCs）凋亡与BMSCs 的MDR1 基因、P-gp 表达的相关性，MDR1 基因过表达或表达沉默对苯所致BMSCs 凋亡的影响，以及NF-κB 信号途径是否参与其中，以期为经遗传修饰后的BMSCs治疗慢性苯中毒提供理论依据。结果显示当HQ浓度可明显抑制BMSCs生长,并呈时间依赖和浓度依赖方式。当HQ浓度为50μM时至125μM可诱导BMSCs凋亡，并呈浓度依赖方式。HQ 以浓度依赖方式抑制BMSCs的MDR1基因和蛋白表达。MDR1过表达体系降低HQ处理对BMSC细胞活力的抑制，降低HQ对BMSC细胞凋亡的诱导作用，MDR1沉默体系升高HQ处理对BMSC细胞活力的抑制，提高HQ对BMSC细胞凋亡的诱导作用。HQ处理下调了NF-κB的核转录活性。HQ抑制了BMSCs 的NF-κB核转录活性。在BMSCs中建立NF-κB p65 过表达体系，相应的MDR1蛋白表达水平上升。NF-κB信号通路抑制剂PDTC在30μm和50 μm时可有效抑制NF-κB p65表达，而且在抑制NF-κB信号通路的同时可下调MDR1基因的mRNA和蛋白表达。通过以上实验结果，我们得出结论：苯可能通过下调MDR1表达产生细胞学毒性，这一机制可能涉及NF-κB信号转导途径。