转化生长因子βⅡ型受体异构体在急性白血病的表达与功能研究

Leukaemia is a clonal haemopoietic disorder characterized by an arrest of the cell maturation and apoptosis and the rapid proliferation and accumulation of undifferentiated cells. The network and balance of cytokines is of major importance in maintaining proper homeostasis of hematopoiesis. Abnormalities in this network may involve in leukemia. TGFβ1 is the strongest known negative regulatory cytokine that controls hematopoietic cell proliferation and induces apoptosis by TGFβ receptor signaling pathway. Dysfunction of the TGFβ signaling pathway can lead to loss of the growth inhibitory effect and thus cell malignant proliferation. In our previous studies, we first reported that the two different mRNA splicing variants of transform growth factor β receptor II (TβRⅡ) existed in acute leukemia cells，while their function are uncertain. Compared with normal bone marrow cells, the ratio of the two spliceosomes is different in acute leukemia cells by semi-RT-PCR.It suggested that the abnormality of TβRⅡisoforms can lead to dysfunction of the TGFβsignaling pathway.To further explore the underlined mechanisms,in the current project, we are going to carry out the following studies: ①CD34 + cells were sorted from acute leukemia cells and normal bone marrow cells by using flowcytometry and then two TβRⅡ isoforms were quantitative detected by quantitative RT-PCR and western blot. ② The vector carrying TβRⅡ isoforms were constructed and then introduced into K562 and HL60 cells expressed lower TβRⅡ. We study the effects of TβRⅡ isoforms overexpressed on the leukemic cell proliferation, differentiation and apoptosis and further explore their mechanism, and Using radio-labelled ligand and receptor binding assay,we study the affinity of two TβRⅡ isoforms with TGFβ1.④We correlate the quantitative expression of two TβRⅡisoforms with clinical phenotype and prognosis. Our findings will provide important insight into the mechanisms of leukemic cell proliferation, differentiation and apoptosis regulated by TGFβ1 signaling ; and the relationship between TβRⅡ isoforms and prognosis will provide guideline to future clinical applications.

白血病的本质特征是白血病细胞过度增殖、分化、凋亡受阻。正负造血细胞因子失衡在其中起重要作用。转化生长因子β(TGFβ)是最强的血细胞增殖抑制因子，其信号通路异常可致TGFβ的抑制作用减弱或丧失,细胞增殖失控。我们在国际上首先发现急性白血病(AL)细胞TGFβⅡ型受体 (TβRⅡ)存在两种mRNA 剪接体，此两种剪接体的功能未见报道。进而用半定量方法检测发现AL细胞与正常相比此两种剪接体含量不同,推测这种异常可能导致TGFβ信号通路的异常。鉴于此，本项目①定量检测CD34+的AL细胞和CD34+正常细胞两种TβRⅡ异构体的含量。②构建含不同TβRⅡ异构体的真核表达载体，转染低表达TβRⅡ基因的K562和HL60细胞，研究两种TβRⅡ异构体对白血病细胞增殖、分化、凋亡的影响及其机制，并检测两种TβRⅡ异构体与TGFβ1亲和力。③研究两种TβRⅡ异构体的表达差异与AL临床表现及预后的关系。

已知转化生长因子β (TGF-β) 信号改变参与白血病的发病过程，尽管在人急性白血病细胞鉴定出新的转化生长因子β的II受体异构体(TβRII-B)，但是其表达模式及异构体功能仍不甚清楚。本项目研究2个TβRII异构体 (TβRII and TβRII-B)在急性白血病表达及其功能。结果表明：急性髓性白血病(AML)存在2个TβRII异构体的异常表达，即在急性髓性白血病细胞主要表达TβRII，而在正常造血干细胞主要表达TβRII-B，进一步功能研究表明相比TβRII，TβRII-B能够促进TGF-β诱导细胞周期阻滞，协同TGF-β促进三氧化二砷诱导的细胞凋亡和维A酸诱导的细胞分化。TβRII-B在促进与TGF-β1的结合及下游蛋白信号的磷酸化及调节靶蛋白的表达水平以及抑制裸鼠体内移植瘤细胞的增殖方面，均强于TβRII。相比之下，TβRII通过抑制TβRII-B阻断维A酸诱导的细胞分化。结合患者临床资料显示，高表达TβRII的AML患者总生存期显著低于低表达TβRII的AML患者。本项目研究结果证明TβRII-B是细胞周期阻滞，细胞凋亡和细胞分化的潜在诱导剂，高表达TβRII的AML患者预后差。