CX43/E-cadherin介导白血病骨髓基质GJIC功能缺失致Ph+ALL残留耐药作用及机制

The effects of treatment on Ph + acute lymphoblastic leukemia(Ph + ALL) is poor.The "asylum"of Hematopoietic microenvironment on leukemia cells is a very important factor in refractory leukemia. Applicants in the preliminary research discovered that the dysfunction of gap junction intercellular communication（GJIC）in leukemia bone marrow stromal cells mediated by Connexin43（CX43）contributes to residual leukemia cells and the decreasing to chemotherapy sensitivity ,but the mechanism is not very clear by now. Since CX43 is closely related to cadherin, this study plans to build a residual disease model in vitro in which the bone marrow stromal cells from Ph + ALL patients are co-cultured with SUP-B15 cells , Using methods of co-immunoprecipitation,siRNA and laser confocal microscopy ect.to detect the Interactions of the complex of CX43/Cadher and the affect to GJIC's function ,to observe the effects to phosphorylation of connexin and Ca2+ flow, to probe into the influence to proliferation and apoptosis of Ph + ALL cells through the BCR / ABL, β-catenin Pathway, and to clarify the dynamic relationship between GJIC function and the residual leukemia mediated by CX43/cadherin. We investigate the study to research the causes and mechanisms of Ph + ALL refractory from a new angle, which may provide some experimental basis for the removal of minimal residual disease and reducing the relapse.

Ph+急性淋巴细胞白血病（Ph+ALL）治疗效果差，造血微环境的"庇护"作用是难以治愈的重要因素。申请者在前期基金课题中发现：连接蛋白43（CX43）介导的白血病骨髓基质间隙连接细胞间通讯（GJIC）功能缺失有助于白血病细胞残留并对化疗药物不敏感，但具体机制不明。CX43发挥作用与钙粘素（E-cadherin）关系密切，本研究拟在体外构建Ph+ALL骨髓基质细胞/SUP-B15细胞共培养残留耐药模型；研究骨髓基质细胞CX43与E-cadherin的相互作用及其对GJIC功能的影响；检测其对连接蛋白磷酸化和Ca+离子交换的作用效应；研究其通过BCR/ABL、β-catenin途径对Ph+ALL细胞增殖、凋亡的特殊影响；阐明由CX43/E-cadherin主导的骨髓基质GJIC功能与Ph+ALL残留耐药间的关系，从一新的角度探索Ph+ALL难治的原因和机制，为清除残留病降低复发奠定实验基础。

Ph＋急性淋巴细胞白血病（Ph＋ALL）治疗效果差，复发率高，究其原因，残留白血病（MRD）是其难以彻底清除和复发的主要原因。造血微环境在正常造血过程中有重要作用，白血病细胞的生长同样依赖造血微环境的调控和支持。骨髓基质细胞（BMSCs）对白血病细胞所产生的支持和庇护作用是白血病发生和难治的重要原因。BMSCs中CX43/E-cadherin 复合体以及CX43介导的GJIC功能与白血病的发生、发展和预后密切相关。本研究构建了体外BMSCs与Ph+ALL细胞共培养残留白血病模型。利用CX43或E-cadherin过表达腺病毒转染上调Ph+ALL患者BMSCs中CX43或E-cadherin的表达，再与ph+白血病细胞SUP-B15细胞体外共培养，检测共培养后的SUP-B15细胞发现：Cx43转染的BMSCs通过直接接触粘附白血病细胞，使白血病细胞凋亡率升高，增殖受到抑制；促进SUP-B15细胞中PI3K和磷酸化GSK-3β的表达，同时抑制β-catenin的表达。构建干扰腺病毒下调正常BMSCs中CX43或E-cadherin的表达，结果显示BMSCs中GJIC功能降低，促进与之共培养的SUP-B15的增殖，抑制其凋亡，促进SUP-B15细胞中PI3K和β-catenin的表达，同时抑制磷酸化GSK-3β的表达，并上调转录因子MMP7、C-MYC、EphB2和FGF20的mRNA表达。同时，在化疗药处理下，下调BMSCs中CX43或E-cadherin的表达，能促进SUP-B15耐药基因的表达，抑制凋亡，促进增殖。进一步研究发现，在骨髓微环境缺氧状态下能诱导CX43表达下降，促进与共培养的白血病细胞增殖，抑制凋亡，降低药物敏感性。上述结果表明：在BMSCs中，CX43能与E-cadherin形成复合体，CX43、E-cadherin的下调能导致GJIC功能缺失，可能通过β-catenin通路进而诱导各白血病相关转录因子表达和耐药基因表达，抑制白血病细胞凋亡，促进其增增殖，上调CX43、E-cadherin的表达，可以逆转白血病细胞恶性程度，促进其凋亡。迄今为止，本课题共发表论著10篇，其中SCI收录6篇；获国家发明专利3项；获2015年重庆市科技进步一等奖1项；第三军医大学新技术二级甲等1项；中国抗癌协会三等奖1项；培养博士研究生1名，硕士研究生6名。