HO-1基因敲除对骨髓微环境介导血液肿瘤耐药的影响及机制研究

The mechanism and reversing strategy for tumor chemoresistance are still being spotlighted at present. This project aimed to explore the effects and mechanism of HO-1 expression in bone marrow microenvironment on hematological malignancies. The results showed us that: 1) Hematological tumor cells derived from clinical samples were insensitive to chemotherapeutic agents as HO-1 was overexpressed in bone marrow stromal cells (BMSCs) detected by real-time PCR and western blot. 2) Co-cultured hematological tumor cells and BMSCs were transfected by lenti-virus-HO-1/siHO-1, the tumor cells cocultured with HO-1 up-regulated the resistance of BMSCs to chemotherapeutic agents. 3) In vivo experiments showed that tumor cells in HO-1+/+ mice were more insensitive to chemothepeutic agents than those in HO-1+/- mice. 4) The drug-resistant mechanism mediated by HO-1 expressed in BMSCs involved in activation of JAK2/STAT5, CXCL12-CXCR4 axis and HIF-1α pathways. Nextly, we’ll verify the pathway induced by tumor microenvironment by second generation sequencing and co-immunoprecipitation, to supply a new strategy to reverse drug-resistance in hematological malignancy.

对肿瘤耐药机制和逆转策略的研究仍是目前热点。本课题旨在研究骨髓微环境中HO-1基因表达对血液肿瘤耐药的影响及机制。结果显示：① Real -time PCR和Western blot检测发现HO-1基因在血液肿瘤患者骨髓基质细胞（BMSCs）中高表达，可导致肿瘤耐药；② 慢病毒介导HO-1/siHO-1感染BMSCs，显示HO-1基因表达水平可影响共培养的肿瘤细胞耐药特性。③采用HO-1+/+，HO-1+/-基因敲除小鼠体内证实：肿瘤细胞在HO-1+/-组的耐药倍数低于HO-1+/+组。④ 通过激动剂及阻断剂方法研究，初步证实其耐药机制是通过JAK2/STAT5、CXCL12-CXCR4轴和HIF-1α等通路激活介导的。在下步研究中我们拟将采用二代测序、免疫共沉淀等在HO-1-/-敲除小鼠体内证实HO-1介导的肿瘤微环境耐药的机制和通路，为逆转肿瘤耐药提供新策略。

耐药是血液恶性肿瘤放化疗失败，死亡率高居不下的重要原因。多年来，研究者从多方面研究并阐释了肿瘤细胞自身因素改变所导致的耐药机制，但至今仍难以突破此瓶颈。本课题旨在研究骨髓微环境中HO-1基因表达对血液肿瘤耐药的影响及机制。结果显示：①HO-1基因在血液肿瘤患者骨髓基质细胞（BMSCs）中高表达，可导致肿瘤耐药；② 慢病毒介导HO-1/siHO-1感染BMSCs，显示HO-1基因表达水平可影响共培养的肿瘤细胞耐药特性。③采用HO-1+/+，HO-1+/-基因敲除小鼠体内证实：肿瘤细胞在HO-1+/-组的耐药倍数低于HO-1+/+组。④通过激动剂及阻断剂方法研究，通过研究我们证实了HO-1 在骨髓微环境中高表达，可以介导恶性血液肿瘤耐药，调控BMSCs中HO-1的表达可以逆转骨髓微环境介导的血液肿瘤耐药；具体探讨的通路机制如下：.1.BMSCs-L-HO-1中CXCL12及受体CXCR4 的表达增加，并激活 CXCL12/CXCR4 轴，磷酸化 PI3K/AKT信号通路，抗凋亡蛋白Bcl-2 表达显著增加，从而使肿瘤细胞产生耐药，肿瘤细胞对 IM 的耐药效应可归因于CXCL12/CXCR4 轴中 HO-1 的特异性激活。.2.我们发现HO-1是APRIL、NF-κB和CCL3的上游基因。HO-1可以减弱MM细胞中硼替佐米对APRIL-NF-κB-CCL3信号传导的抑制作用，如果HO‐1和APRIL同时被抑制，可能会减少患者的骨质破坏，也会降低MM患者的耐药风险。.3. 骨髓基质细胞中HO-1过表达通过激活P13K/AKT磷酸化促进VEGF的分泌，通过VEGF作用于共培养的Super B15和CCRF-SB 细胞，从而诱导B-ALL细胞对长春新碱的耐药。.以上研究为研发靶点抑制剂或信号通路阻断剂提供理论依据，为逆转肿瘤耐药提供新策略。