Experimental Study On The Biological Characteristics And Gene Expression Profile Of The Elastic Phase Of Chronic Myelogenous Leukemia Bone Marrow Mesenchymal Stem Cells

Objective:Chronic myeloid leukemia (CML) is a myeloproliferative disease originating in multipotent HSCs which acquire the reciprocal translocation t(9;22)(q34;q11) characterized cytogenetically by the presence of the Philadelphia (Ph1) chromosome, and forming a hybrid bcr-abl gene that codes for the BCR-ABL oncoprotein. CML is a myeloproliferative neoplasma originated from multipotent hematopoietic stem cells. CML usually presents in chronic phase, and CML progresses from a chronic phase to an accelerated phase, followed by a terminal blastic phase of lymphoid or myeloid phenotype.CML in blast crisis phase (CML-BP) is highly refractory to the current standard induction chemotherapies. There are several treatment options for CML. The only known curative therapy for CML is stem cell transplantation, but the high transplant-related morbidity from graft-versus-host disease and the high mortality rate of10%to20%have greatly restricted its use. Molecularly targeted therapy includes imatinib, which inhibits the BCR-ABL tyrosine kinase, really prolonged the survive of CML patients and decreased morbidity and mortality rates. However, several clinical trials have shown that disease relapsed when patients withdrawhand, some patients developed primary or secondary resistance to imatinib, especially in blast crisis. So, the treatment of CML blast crisis is disappointing, blast crisis is highly resistant to treatment.At present, most of the research has focused on hematopoietic stem cell abnormalities, such as found in most blast crisis patients have BCR/ABL fusion gene abnormalities, and is often accompanied by other genetic abnormalities, such as tumor suppressor genes p53, p16and Rb, cancer gene RAS, mye and transforming gene Evi-1. But people pay little attention for the significance of the bone marrow mesenchymal stem cells (BMMSCs) in chronic myeloid leukemia blast crisis phase.BMMSCs are present in the bone marrow, which are different from other bone marrow stem cells, BMMSCs were first discovered by Friedenstein in1968, BMMSCs have the ability of self-renewal and multilineage differentiation capacity. Under the certain conditions, BMMSCs can differentiate into a variety of cells such as osteoblasts, adipocytes, chondrocytes, myocytes, nerve cells, and secrete various cytokines, and participate in the hematopoietic microenvironment. Through direct contact with hematopoietic cells and expression of a variety of cytokines, BMMSCs can affect the proliferation and differentiation of hematopoietic cells.The reason of CML blast crisis was unclear, but some people found the majority of tumor cells obtain a super unlimited ability to add value due to the abnormal activation of the signaling pathway. From this perspective, we think if there are some difference functions between CML blast crisis phase BMMSCs and CML-Cp-BMMSCs. This issue needs further exploration and research. In this study, we observed CML blast crisis phase BMMSCs biological characteristics, and we studied the similarities and differences between CML blast crisis phase BMMSCs and CML-Cp-BMMSCs, such as proliferation, differentiation, apoptosis and other aspects. We made K562cells and CML blast crisis phase primary cells respectively co-cultured with CML blast crisis phase BMMSCs, contrast to CML-CP and normal BMMSCs, observed the changes of proliferation and apoptosis and detected the possible mechanisms. Finally, by the study of gene chip, we have observed CML blast crisis phase BMMSCs gene expression characteristics and screened out key difference genes and signaling pathways. This study is aimed at determining changes in gene expression of CML blast crisis phase BMMSCs that occur in the evolution of the chronic phase to blast crisis phase.Methods:1. Isolation and cultivation and expand of CML chronic phase and blast crisis phase BMMSCs from patients, normal BMMSCs as control group. Observed the primary cells culture characteristics and amplification characteristics of blast crisis BMMSCs. BMMSCs aspirates with heparin anticoagulation was taken from CML patients. The cells were inoculated in25m2cell culture flask, cultured at37℃with5%CO2and saturated humidity.2. The morphous of cells was observed and record under the inverted microscope:we observed the morphological of BMMSCs, selected the cells that grow well after the P3-generation. The slides covered by BMMSCs were stained by wrigh’s-Giema’s staining, fixed and observed morphology under the inverted microscope and photographed.3. The immunophenotype of MSCs was detected by flow cytometer in CML blast crisis phase, chronic phase and normal group.4. MTT method for the determination of CML-BP-BMMSCs growth curve characteristics, we observed the amount of colony formation (CFU-F) changes.5. Flow cytometric method for the detection of blast crisis BMMSCs of the cell cycle distribution and spontaneous apoptosis.6. RT-PCR detection of blast crisis phase BMMSCs expression of hematopoietic growth factor (SCF, IL-12, TPO, IL-6, GM-CSF) and adipogenic differentiation marker gene, and whether difference or not from chronic phase of BMMSCs.7. Under the certain conditions, BMMSCs can differentiate into a variety of cells, normal BMMSCs as control group.8. we studied the gene expression characteristics of CML blast crisis BMMSCs, analysed the microarray data to understand the differences of gene expression and signal pathway between CML blast crisis phase and chronic phase BMSCs. Then we detected and analysed the accuracy of identification of gene chip by real time fluorescent quantitative PCR. And we would study the correlation between the differentially expressed gene and CML blast crisis.9. BMMSCs were co-cultured with K562cells or CML-BP Primary cells. Cell proliferation, cell viability of BMMSCs on K562cells or primary cell on K562cells was detected by MTT assay and direct counting method, as well as BMMSCs on K562cells and primary cell treated by adriamycin.10. The apoptosis rate was measured by flow cytometry. Experiment Groups:①control group:K562cells or primary cells+ADM;②The Nc-BMMSCs+ADM Group;③The CML-Cp-BMMSCs+ADM group;④The CML-Bp-BMMSCs+ADM group;⑤The Bp-BMMSCs+ADM+recombinant human DKK1protein group.11. Western blot (WB) detection of apoptosis related proteins:the protein level changes of activation of Caspase-3, BCL-2, Bax and β-Catenin.Results:1. Primary CML blast crisis phase BMMSCs growth were slow, the clone formation of adherent cells were less. But in P3-generation and later, cell proliferation ability enhanced, basically consistent with CML chronic phase BMMSCs group and normal control group.2. Cell growth curve display CML blast crisis phase BMMSCs has a latency period, growth period, platform stage growth characteristics.3. The formation of CFU-F capacity of Primary CML blast crisis is diminished than of chronic and normal controls, the latter two had no obvious difference.4. Spontaneous apoptosis of CML blast crisis phase BMMSCs was6.54±1.08%, and G0/G1was89.92±0.89%. There was no statistically significant compared with BMMSCs of chronic phase and the normal group. Multiplex differentiation capacity experiment indicated BP-BMMSCs could differentiated into adipocytes and osteoblasts, and RT-PCR could detect the differentiation marker genes, then compared with CP-BMMSCs group and normal group, there was no obvious difference.5. The detection of cytokines expressionBoth of CML blast crisis phase and the chronic phase BMMSCs could not express bcr/abl fusion gene. IL-6, SCF, TPO, G-CSF and IL-12have detected between the two phase BMMSCs, but the expression of IL-6, IL-12, SCF and TPO in BP-BMMSCs was down-regulated. 6. The difference between the two groups genes6.1The abundance of gene expression change2times is significant. Comparison of CML blast crisis BMMSCs and chronic phase of BMMSCs gene expression profiles, we received a total of558differential genes, of which289up-regulated genes and269down-regulated genes.6.2Pathway analysisThrough the analysis, we screened out signal transduction pathways and there was a significant difference between CML blast crisis group and chronic phase f BMMSCs group. The microarray results showed a total of98signal transduction pathways changed, changed genes in the pathway of not less than4, and with P value<0.01for the standard, we screened out of15signal pathways which changed significantly. This15signal pathways include cell adhesion pathways, cell apoptosis pathway, inflammation related pathways, cell structure and integrity related pathway.6.3GO analysisThe chip GO analysis of CML blast crisis phase BMMSCs group and chronic phase BMMSCs group has revealed, in biological processes, there were298of cellular processes related differential expressed genes, other higher proportion genes involved cellular component organization or biogenesis at cellular level; cell adhesion; biological adhesion; G-protein coupled receptor protein signaling pathway; cell junction assembly; calcium-independent cell-cell adhesion; glycogen metabolic process and regulation of osteoblast differentiation.6.4Verification of partial differential expression genesIn order to verify the differentially expressed genes, we selected4differential expressed genes. Real time quantitative PCR display:gene chip and PCR results were consistent; it proved the microarray results are reliable.7.The affect of BMMSCs on K562cells and CML blast crisis primary cell proliferation7.1The affect of BMMSCs on K562cells and CML blast crisis primary cell. CML blast crisis phase BMMSCs, Cp-BMMSCs and normal BMMSCs were co-cultured with K562cells, could inhibit the growth of K562cells and primary cells(p<0.05). The inhibition of CML-Bp-BMMSCs was not significant.7.2K562cells separate training group to join doxorubicin48hours after the cells vigor significantly dropped to73.13±2.42%, and blast crisis BMMSCs trained group the cell vitality for83.78±5.17%, significantly (P<0.05). The normal group BMMSCs and chronic phase respectively K562cell in BMMSCs trained, testing the cell vitality are75.45±3.27%and77.56±3.11%(P>0.05).The normal group BMMSCs, CML-CP-BMMSCs and CML blast crisis phase BMMSCs Were co-cultured with CML primary cell of CML-BP, combined with adriamycin in treatment, the cell viability of three groups respectively was51.47±3.22%,56.36±2.39%and65.69±3.24%, the difference was significant (P<0.05). Comparison of the three groups, the cell viability of BP-BMMSCs co-cultured group was strongest (P<0.05), normal BMMSCs and chronic phase BMMSCs co-culture group comparison no difference (P>0.05).8. CML blast crisis phase BMMSCs could protect K562cells to reduce doxorubicin induced apoptosisThe apoptosis rate of adriamycin treatment of K562cells cultured alone was23.1±2.45%, and the three co-cultured groups of normal BMMSCs, CML-CP-BMMSCs and BP-BMMSCs with K562cells, the apoptosis rates of adriamycin treatment were19.9±0.82%,18.5±1.63%and13.4±2.15%(P<0.05). Among the three groups, the CML-BP-BMMSCs had the strongest protective effect(P<0.05). The latter two culture groups were no significant difference(P>0.05). The same results to CML-BP primary cells.9. The detection of apoptosis pathway associated proteins:After adriamycin treated, BCL-2protein level was down-regulated, Bax protein level and the activation of Caspase-3protein level were up-regulated. BCL-2/Bax ratio was up-regulated. After co-cultured with CML-blast crisis phase BMMSCs, BCL-2protein was up-regulated again, howerve, Bax protein and the activation of Caspase-3protein level were down-regulated. Blast crisis phase BMMSCs could decrease the role of ADM induced cell apoptosis by regulated the expression of apoptosis pathway related protein. The CML chronic phase BMMSC co-cultured group also appeared similar change, but weaker.10.Neutralizing antibody to reversal BMMSC protective effect on K562cellsThis experiment observed CML blast crisis phase BMMSCs antagonized K562cells apoptosis induced by ADM, after treated with DKK1neutralizing antibody, partial reversed of blast crisis phase BMMSCs protective effect, led to K562cells apoptosis rate increased (13.4±2.15%Vs.16.3±1.67%, P<0.05).Conclusions:1. Primary CML blast crisis phase BMMSCs growth were slow, the clone formation of adherent cells were less. But in P3-generation and later, cell proliferation ability enhanced, basically consistent with CML chronic phase BMMSCs group and normal control group.CML blast crisis of primary BMMSCs slow growth, Colony formation reducing, fusion time extension. And CFU-F forming ability is abate compare with a chronic phase BMMSCs and normal controls the latter two had no obvious difference. But after the successful passage to the P3generations, cell proliferation ability enhancement, and CML in chronic phase BMMSCs and normal control group comparison of basically were same, with the growth characteristics of incubation period, growth period, platform stage.2. CML blast crisis phase BMMSCs high expressed mesenchymal stem cell surface markers CD44, CD90and CD106, low expression stem cell marker CD34and CD45, CML blast crisis phase BMMSCs conformed to mesenchymal stem cell immunological characteristics. CML blast crisis phase BMMSCs could differentiated into adipocytes and osteoblasts, spontaneous apoptosis6.54±1.08%, G0/G1was89.92±0.89%, there was no statistically significant compared with BMMSCs of CML chronic phase and the normal groups. 3. CML blast crisis phase BMMSCs and CP-BMMSCs did not express BCR/abl fusion gene, IL-6, SCF, TPO, G-CSF and IL-12have detected between the two phase BMMSCs, but the expression of IL-6, IL-12, SCF and TPO in BP-BMMSCs was down-regulated.4. In this part we detected the differences of CML blast crisis and chronic phase of BMSCs gene expression profile, find out differential expressed genes and a number of signal transduction pathways between the two groups, enabled us to understand CML blast crisis and chronic phase BMSCs certainly exist differences on gene expression. Whether there is a relationship between the bone marrow microenvironment represented by BMMSCs and CML blastic. We still need to do a large number of research.5. Comparison of the three groups, the cell viability of BP-BMMSCs co-cultured group was strongest (P<0.05), normal BMMSCs and chronic phase BMMSCs co-cultured group comparison no difference (P>0.05), both of Chronic phase BMMSCs and normal BMMSCs group can inhibit K562cell and primary cell proliferation. Blast crisis BMMSCs could partially antagonized K562cells and CML blast crisis phase primary cell proliferation inhibition reduced by adriamycin.6. CML blast crisis phase BMMSCs antagonized adriamycin induced apoptosis of K562cells, compared with the CML chronic phase and normal BMMSCs, the CML blast crisis phase BMMSCs had the strongest protective effect(P<0.05). The latter two culture groups were no significant difference(P>0.05).7. CML blast crisis phase BMMSCs through regulated apoptosis pathway related protein expression, for example the up-regulation of BCL-2, down-regulation of Bax and activated Caspase-3protein levels, and activated the Wnt pathway of K562cells, antagonized adriamycin induces apoptosis, This experiment observed CML blast crisis phase BMMSCs antagonized K562cells apoptosis induced by ADM, after treated with DKK1neutralizing antibody, partial reversed of blast crisis phase BMMSCs protective effect, led to K562cells apoptosis rate increased.