【作者】 李招权；

【导师】 邹全明；

【作者基本信息】 第三军医大学 ， 临床检测诊断学， 2008， 博士

【摘要】 研究目的Wnt5a是Wnt蛋白家族重要成员之一,不仅与个体发育、细胞粘附、迁移、增殖、分化、极性和死亡等相关,甚至与肿瘤发生关系密切。目前研究发现,Wnt5a在肿瘤中既有增高表达又有下调表达,它既可能激活Wnt/β-catenin信号传导途径,又可能激活非经典Wnt5a/Ca²⁺信号传导途径,故Wnt5a既可能是癌基因,又可能是抑癌基因。关于Wnt5a在血液病中表达及其作用的研究鲜见报道,更罕见外源性Wnt5a对白血病细胞有何生物学效应的研究报道。为此,本研究首先检测了Wnt5a及β-catenin在一些血液病病例及白血病细胞株中的表达情况,在此基础上,以K562细胞为细胞模型,观察外源性Wnt5a是否激活非经典Wnt5a/Ca²⁺信号传导途径,并观察其生物学效应,以探讨Wnt5a在白血病发生中的作用和机制,为白血病的治疗寻求新的治疗靶点。研究方法1.应用RT-PCR方法检测31例各类血液病病例样本和3种白血病细胞株Wnt5a表达,并检测11例髓细胞白血病病例样本和3种白血病细胞株β-catenin的表达。2.应用HEK293细胞扩增带有标记基因GFP的重组腺病毒AdWnt5a和AdGFP,以AdWnt5a和AdGFP分别感染CHO细胞,制备Wnt5a和GFP条件培养液,以western blot鉴定条件培养液Wnt5a蛋白的表达。3.以荧光染料Fluo-3/AM预染K562细胞,以激光共聚焦显微镜分别观察Wnt5a和GFP条件培养液对K562细胞Ca²⁺内流的影响,观察外源性Wnt5a是否激活K562细胞非经典Wnt5a/Ca²⁺信号传导途径。4.分别以Wnt5a和GFP条件培养液培养K562细胞1⁵ d,以RT-PCR、免疫细胞化学和western blot等方法观察K562细胞β-catenin和cyclin D1的表达变化,观察外源性Wnt5a与Wnt/β-catenin信号传导途径的作用关系。5.分别以Wnt5a和GFP条件培养液培养K562细胞1⁵ d,观察外源性Wnt5a对K562细胞的生物学效应:每天计数细胞数量,绘制生长曲线,观察外源性Wnt5a对细胞增殖的影响;以流式细胞仪分析细胞周期分布,观察外源性Wnt5a对细胞周期分布的影响;以DNA Ladder电泳和AO/EB染色法,观察外源性Wnt5a对细胞凋亡的影响;细胞以瑞氏染液染色,光镜下观察细胞形态学改变,并利用免疫细胞化学染色检测细胞表面分化抗原CD13、CD41、CD68和GlyA的表达变化,观察外源性Wnt5a对细胞分化的影响。实验结果1.Wnt5a在髓细胞白血病病例样本及K562、HL-60细胞中表达缺失,而β-catenin在多数髓细胞白血病病例样本及K562细胞和Jurkat细胞呈高表达。2.分别利用重组腺病毒AdWnt5a、AdGFP感染CHO细胞,成功制备Wnt5a及GFP条件培养液,经western blot鉴定Wnt5a条件培养液含Wnt5a蛋白,而GFP条件培养液无表达。3.GFP条件培养液不能促进K562细胞Ca²⁺内流,而Wnt5a条件培养液可促进K562细胞Ca²⁺内流,且这种作用可被Wnt5a抗体抑制。4.Wnt5a条件培养液培养不能下调K562细胞β-catenin mRNA的表达,但能下调K562细胞β-catenin及cyclin D1蛋白的表达。5.Wnt5a条件培养液对K562细胞的生物学效应有:细胞增殖明显受抑制;细胞周期分布G1期细胞比例增多,S期及G2期细胞比例减少;细胞形态出现了向成熟细胞分化的特征,细胞表面分化抗原CD13、CD41及GlyA表达变化不明显,CD68表达阳性率显著升高;细胞DNA Ladder电泳未出现明显梯形条带,激光共聚焦显微镜观察细胞凋亡率无明显变化。研究结论1.Wnt5a表达缺失和β-catenin高表达可能是髓细胞白血病的发生相关因素。2.外源性Wnt5a可激活K562细胞非经典Wnt5a/Ca²⁺信号传导途径,并在蛋白水平下调K562细胞β-catenin及cyclin D1表达,提示在K562细胞中,外源性Wnt5a可抑制Wnt/β-catenin信号传导途径。3.外源性Wnt5a可使K562细胞发生G1期阻滞,抑制K562细胞的增殖,诱导K562细胞向单核细胞分化,但无诱导K562细胞凋亡作用。更多还原

【Abstract】 ObjectiveWnt5a, a family member of the Wnt proteins, plays important roles in body development, cell adhesion, motility, proliferation, differentiation, polarity and death, even was proposed to be close relative to tumorigenesis. Nowadays, studies indicates Wnt5a, which can activate the Wnt/β-catenin signaling pathway or noncanonical Wnt5a/Ca²⁺ signaling pathway, is overexpressed in some tumors and also suppressed in other ones. So, Wnt5a maybe is both oncogene and antioncogene. We found rare studies on Wnt5a’s expression and it’s function in hematopoietic disease, and haven’t found report about what effect exogenous Wnt5a would contribute to leukemic cells. Therefor, we detected the expression of Wnt5a andβ-catenin in hematopoietic disease samples and leukemic cell lines, and choiced K562 cell lines as research model to observe whether exogenous Wnt5a could activate noncanonical Wnt5a/Ca²⁺ signaling pathway and it’s biologic effects. Through these studies, we try to uncover the function and mechanism of Wnt5a in leukemiagenesis and to seek new target for leukemia therapy.Methods1. The expression of Wnt5a in 31 hematopoietic disease samples and 3 kinds of leukemic cell lines, and that ofβ-catenin in 11 myeloid leukemic samples and 3 kinds of leukemic cell lines, were detected by RT-PCR.2. Amplification of AdWnt5a and AdGFP （both labelled with GFP tag gene） were prepared by infecting HEK293 cells respectively. The Wnt5a and GFP condition medium were separately prepared by collecting the culture supernatants medium of CHO cells infected with AdWnt5a and AdGFP. The expression of Wnt5a protein in condition medium were identified by western blot.3. To observe whether exogenous Wnt5a could activate noncanonical Wnt5a/Ca²⁺ signaling pathway, K562 cells were previously stained with fluorescence dyestuff Fluo-3/AM, then the influence on Ca²⁺ inflow in K562 cells treated with Wnt5a and GFP condition medium were checked with laser confocal scanning microscope.4. To observe the relationship between exogenous Wnt5a and Wnt/β-catenin signaling pathway, K562 cells were separately treated with Wnt5a and GFP condition medium for 1 to 5 days, then the expression ofβ-catenin and cyclin D1 in K562 cells were detected by RT-PCR, immunochemistical staining and western blot respectively.5. Separately treated with Wnt5a and GFP condition medium for 1 to 5 days after, the biologic effects of exogenous Wnt5a on K562 cells were observed as follow: counting cells number each day and figuring out proliferation curve to observe the influence of exogenous Wnt5a on cell proliferation; detecting the cell cycle distribution of cells by flow cytometry to observe the influence of exogenous Wnt5a on cell cycle; observing the influence of exogenous Wnt5a on cell apoptosis by DNA Ladder electrophoresis and with laser confocal scanning microscope after AO/EB staining; observing cellular morphologic changes with light microscope after Wright’s staining and detecting the expression changes of CD13, CD41, CD68 and GlyA by immunochemistical staining to observe the influence of exogenous Wnt5a on cell differentiation.Results1. Wnt5a expression was lost in myeloid leukemic samples and K562, HL-60 cells, butβ-catenin were overexpressed in most myeloid leukemic samples, K562 cells and Jurkat cells.2. The Wnt5a and GFP condition medium were successfully prepared with AdWnt5a, AdGFP to infect CHO cells separately. Identified by western blot, Wnt5a protein was found expressed in Wnt5a condition medium but not in GFP condition medium.3. GFP condition medium couldn’t stimulate K562 cells Ca²⁺ inflow but Wnt5a condition medium could, and the Ca²⁺ inflow could be blocked with Wnt5a antibody.4. Wnt5a condition medium did not down-regulate the expression ofβ-catenin mRNA, but suppressed the expression ofβ-catenin and cyclin D1 protein in K562 cells.5. The biologic effects of Wnt5a condition medium on K562 cells: cell proliferation was significantly inhibit; the cells number of G1 phase increased and that of S phase and G2 phase decreased; mature morphologic differentiational features was detected and the positive rate of CD68 significantly increased, but that of CD13, CD41 and GlyA had no obvious changes; no obvious trapeziform band was detected in DNA Ladder electrophoresis and apoptosis rate didn’t obviously rise observed with laser confocal scanning microscope.Conclusions1. The loss of Wnt5a expression and overexpression ofβ-catenin may be is relative to leukemiagenesis.2. Exogenous Wnt5a can activate the noncanonical Wnt5a/Ca²⁺ signaling pathway and downregulated the expression ofβ-catenin and cyclin D1 proteins in K562 cells. It indicate that exogenous Wnt5a can inhibit Wnt/β-catenin signaling pathway in K562 cells.3. Exogenous Wnt5a can induce K562 cells arise G1 phase arrest, inhibit K562 cells proliferation, and induce K562 cells differentiate to monocyte, but can’t induce K562 cells apoptosis.更多还原