Effect Of B-ALL Cells On The Differentiation Of Bone Marrow Mesenchymal Stem Cells

PART ONE EXPRESSION OF NOTCH SIGNALING RELATED LIGAND JAGGED1 IN CHILDREN WITH ACUTE LEUKEMIAObjective:To investigate expression characteristics and the clinical significance of Notch signaling related ligand Jagged1 in acute leukemia children.Methods:Bone marrow isolated from 88 cases of childhood acute leukemia patients were divided into 3 groups:AML group, B-ALL group, and T-ALL group. Bone marrow isolated from 18 cases of childhood ITP patients were set as control. And B-ALL was divided into standard risk, middle risk, and high risk. Real-Time PCR was utilized to detect the expression of Jaggedl RNA in bone marrow mononuclear cells.Results:1) The expression of Jaggedl could be detected in all samples from AML group, group B-ALL, T-ALL group and the control group;2) The expression levels of Jaggedl was higher in AML group than the control group, but there was no statistically significant difference (P>0.05, n= 25);3) The expression levels of Jaggedl was higher in B-ALL group than the control group, and the difference was statistically significant (P< 0.05, n= 52);4) The expression levels of Jaggedl was lower in T-ALL group than the control group, and the difference was statistically significant (P< 0.05, n=11);5)In B-ALL groups, the expression levels of Jaggedl in high risk group was significantly higher than that in standard risk and middle risk group (P<0.05), respectively.Conclusion:The expression levels of Jaggedl is inconsistent in different types of children acute leukemia. Compared with the control group, B-ALL has high expression, T-ALL show lower expression, and AML has normal expression, indicate that the expression of Jaggedl is abnormal in ALL.PART TWO EFFECT OF B-ALL CELLS ON THE OSTEOGENIC DIFFERENTIATION OF BONE MARROW MESENCHYMAL STEM CELLS AND ITS MECHANISMObjective:To investigate the effect of B-ALL cells on the osteogenic differentiation of BMSCs and the role of Notch signaling in this process.Methods:We cocultured BMSCs with B-ALL cells in osteogenic induction medium. BMSCs cultured alone and BMSCs cocultured with Bone marrow mononuclear cells were set as control. The expression levels of Notch 1, Hes1, and the osteogenic markers Runx2, Osteopontin (OPN), and Osteocalcin (OCN) were assessed by real time PCR and western blotting on day 3. Alkaline phosphatase (ALP) activity was analyzed using an ALP kit, and mineralization deposits were detected by Alizarin red S staining on day 14. And then we treated BMSCs with Jagged1 protein and anti-Jaggedl neutralizing Ab. The expression of Notchl, Hesl, and the abovementioned osteogenic differentiation markers was measured.Results:1) Compared with control group, the expression level of OPN, OCN in BMSCs was lower in B-ALL cells coculture group, and ALP activity and mineralization ability was reduced;2) In BMSCs and B-ALL cells coculture group, the expression level of Notch signaling downstream gene Hesl was elevated, which indicate that the Notch pathway was activated in BMSCs;3) Anti-Jaggedl-Ab neutralizing antibodies can inhibit Notch signaling in BMSCs, and the expression of OPN, OCN in BMSCs increased at the same time;4) Recombinant protein Jagged1 can activate the Notch signaling in BMSCs, and the expression of OPN, OCN decreased at the same time;5) Anti-Jaggedl-Ab neutralizing antibodies can decrease the expression of Hesl, but increased the expression of Runx2 in BMSCs.Conclusion:ALL cells could inhibit the osteogenic differentiation of BMSCs. The possible mechanism is B-ALL cells can activate Notch signaling in BMSCs by Jaggedl, and its downstream gene Hes1 reduced osteogenesis differentiation transcription factor Runx2 protein expression levels, thus inhibit osteoblast differentiation of BMSCs.PART THREE EFFECT OF B-ALL CELLS ON THE ENDOTHELIAL DIFFERENTIATION OF BONE MARROW MESENCHYMAL STEM CELLS AND ITS MECHANISMObjective:To investigate the effect of B-ALL cells on the endothelial differentiation of BMSCs, and explore the possible mechanism in the process.Methods:We cocultured BMSCs with B-ALL cells. BMSCs cultured alone and BMSCs cocultured with Bone marrow mononuclear cells were set as control. The mRNA expression levels of Notchl, Hesl and the endothelial markers Flk-1、CD31、vWF were assessed by real-time PCR, and the protein expression levels of Flk-1、CD31 were detected by western blotting on day 3 or day 7. And then we treated BMSCs with anti-Jagged1 neutralizing Ab. The expression of Notchl, Hes1, and the abovementioned endothelial differentiation markers was measured. At last, we detect VEGF secretion protein levels in B-ALL and BMSCs coculture system.Results:1) At 3 days, the mRNA expression level of CD31, vWF, Flk-1 and the protein expression levels of Flk-1, CD31 in BMSCs were higher than the control group, but there were no significant difference (P> 0.05); 2) At 7 days, the mRNA expression level of CD31, vWF, Flk-1 and the protein expression levels of Flk-1, CD31 in BMSCs were higher than the control group, and there were significant differences (P< 0.05); 3) In BMSCs and B-ALL cells coculture group, the expression level of Notch signaling downstream gene Hes1 was elevated, which indicate that the Notch pathway was activated in BMSCs.4) Anti-Jaggedl-Ab neutralizing antibodies can inhibit Notch signaling in BMSCs, but the mRNA expression levels of Flk-1, CD31, vWF and the protein expression levels of Flk-1, CD31 did not change between groups.5) The secretion protein expression levels of VEGF was elevated in co-culture groups, and the expression levels of VEGF in B-ALL cells and BMSCs were corresponding elevated.Conclusion:B-ALL cells could promote BMSCs differentiate into endothelial cell, and its possible mechanism is that the VEGF secretion protein levels is elevated in B-ALL and BMSCs coculture system, and the Notch signaling do not play a main role in this process.