Preliminary Study Of Wnt Signaling Pathway In Chronic Myeloid Leukemia

Chronic myeloid leukemia is a place in the pluripotent hematopoietic stem disease of malignant proliferation of bone marrow cells, can detect the characteristic BCR-ABL fusion gene, disease by chronic period gradually progress to acute phase, the prognosis is poor. At present, conventional therapy still not completely cured, the existence of leukemia stem cell and drug resistance of leukemia cells is a important reason. Wnt signaling pathway regulate the normal cell growth and differentiation, it was abnormal activated in a variety of hematological malignancies and tumor stem cells, abnormal activated of wnt signaling pathway has close relationship with disease progression、durg resistance. Study on the relationship of the wnt signaling pathways and CML,which has important theoretical and practical significance.Objective:Test the expression of WNT signaling molecules and related in different diseased cells and different disease states of chronic myeloid leukemia. Study the expression level of wnt-1、wnt inhibitory factor-1、β-catenin、cyclinD1in K562、Sorting CD34positive cells、 chronic phase and blastic crisis patients with chronic myeloid leukemia. Whether there exist differences in CD34positive cells and patients with chronic and blastic phase between the expression analysis of Wnt signal and its related factors.Know the relationship between Wnt signaling pathway and disease progression and drug resistance. Explore the new target spot of treatment with chronic myeloid leukemia.Method:K562cell culture, Immunomagnetic separation of CD34positive cells, flow cytometry test the expression of CD34antigen and cell cycle of K562and separation of positive cells. Through methyl cellulose clone culture,cultivate K562and CD34positive cells, compare the colony forming ability with K562and CD34positive cells. RT-PCR test the expression level of wnt-1、wnt inhibitory factor-1、β-catenin、cyclinD1、 ABCG2in K562and separation of positive cells. Collect the bone marrow sample of patients with chronic and blastic phase of chronic myeloid leukemia and patients with benign hematological diseases. Draw RNA, Real-time RT-PCR was applied to compare the mRNA expression of the wnt-1,wnt inhibitory factor-1,β-catenin and cyclinDl in patients with chronic and blastic phase of chronic myeloid leukemia and patients with benign hematological diseases. SPSS17.0statistical analysis the transcriptional level of wnt-1、wnt inhibitory factor-1、β-catenin、 cyclinDl mRNA in patients with chronic phase and blast crisis of chronic myeloid leukemia and patients with benign hematological diseases. We use the nonparametric Mann-Whitney-U rank sum test in between three group comparison. Between each target gene correlation analysis use the spearman rank correlation analysis.Result:Flow cytometric analysis showed that the proportion of cells expressing CD34antigens of0.8% in K562, the percentage of cells in G0/G1phase is33.7%, the percentage of cells in S phase is44.2%, the percentage of cells in G2/M phase is22.1; the proportion of cells expressing CD34antigens of79.2% in sort positive cell, the percentage of cells in G0/G1phase is55,5%, the percentage of cells in S phase is9.4%, the percentage of cells in G2/M phase is35%. Through methyl cellulose clone culture, cell colony formation counts of K562is13.67+10,97, cell colony formation counts of sort positive cell is79.50±18.96,the expression levels of wnt-1、 wnt inhibitory factor-1、β-catenin、cyclinD1、ABCG2in K562separately is49.30×10-2±12.10×10-2、0.087×10-2±0.0130×10-2,150.84±9.48、20.08±0.61、0.22×10-2±0.049×10-2; the expression levels of wnt-1、wnt inhibitory factor-1、β-catenin、cyclinD1、ABCG2in sort positive cell separately is332.11X10"2±51.71×10-2.0.027×10-2±0.010×10-2、187.21±16.22、25.64±2.54、2.6×10-2±1.1×1×10-2. the expression level of wnt-1、wnt inhibitory factor-1、β-catenin、cyclinDl in patients with chronic myeloid leukemia blast phase separately is30.55×10-2、0.06×10-2、153.05、18.19, the expression level of wnt-1、wnt inhibitory factor-1、β-catenin、 cyclinDl in patients with chronic myeloid leukemia chronic phase separately is3.02X×10-2、1.81×10-2、36.53、1.21, the expression level of wnt-1、Wnt inhibitory factor-1、β-catenin、cyclinDl in patients with benign blood disease separately is0.33×10-2、19.67×10-2、21.68、0.19, we use the nonparametric Mann-Whitney-U rank sum test the target gene in between three group comparison, the expression level of wnt-K wnt inhibitory factor-1、βcatenin、cyclinDl in between the three groups exist the difference, P<0.05, differences are statistically significant; Colony forming ability and expression level of ABCG2in sort positive cell are higher than K562, the expression level of wnt-1、β-catenin、cyclinDl in sort CD34positive cell are higher than patients with chronic myeloid leukemia blastic crisis,blastic crisis patient are higher than chronic phase patient, chronic phase patient are higher than benign blood disease patient.the expression level of wnt inhibitory factor-1in sort positive cell is lower than blastic crisis patient, blastic crisis patient is lower chronic phase patient, chronic phase patient is lower than benign blood disease patient. Correlation analysis adopt the nonparametric spearman rank correlation, the expression level of wnt-1and β-catenin is significant positive correlation in chronic phase、 blast phase and benign blood disease patient(r=0.993, p<0.05, r=1, r=0.991, p<0.05). the expression level of wnt-1and β-catenin is perfect positive correlation in blast phase patient, the expression level of wnt-1and cyclinDl is perfect positive correlation in chronic phase, blast phase and benign blood disease patient (r=1, r=1, r=1). the expression level of β-catenin and cyclinDl is significant positive correlation in chronic phase, blast phase and benign blood disease patient (r=0.993, p<0.05, r=1, r=0.991, p<0.05), the expression level of β-catenin and cyclinDl is perfect positive correlation in blast phase patient, the expression level of wnt inhibitory factor-1and wnt-1is significant negative correlation in chronic phase、blast phase and benign blood disease patient(r=-0.993、 p<0.05, r=-0.988、 p<0.05, r=-0.991、p<0.05), the expression level of wnt inhibitory factor-1and β-catenin is significant negative correlation in chronic phase、blast phase and benign blood disease patient (r＝-0.986、p<0.05, r=-0.988、p<0.05, r=-0.982、p<0.05), the expression level of wnt inhibitory factor-1and cyclinDl is significant negative correlation in chronic phase、blast phase and benign blood disease patient (r=-0.993、p<0.05, r=-0.988、p<0.05, r=-0.991、p<0.05).Conclusion:Chronic myeloid leukemia as a malignant blood disease, he matopoietic cell proliferation and differentiation disorder, there are a few CD34positive leukemia stem cells in chronic myeloid leukemia.a bnormal activation of wnt signal pathway in patient with blastic cris is of chronic myeloid leukemia and CD34positive cells. It pointed ou t that there is maybe a correlation in between disease progression an d excessive activation of wnt signaling pathway. The activation of wn t signaling pathway in CD34positive cells abnormally higher than pat hological cell of blastic crisis and chronic phase. It is a reason tha t progression and refractoriness of chronic myeloid leukemia. Excessi ve activation of wnt signal pathway maybe is a important factor of th e disease progression and drug resistance.