| Background: Leukemia is a kind of malignant cloned disease of hematopoietic stem cells.Clonal leukemia cells proliferate in bone marrow and other hematopoietic tissues because of the mechanisms of proliferation out of control,disorder of differentiation,apoptosis and infiltrate other non hematopoietic tissues and organs.Inhibiting normal hematopoiesis.It is reported that the incidence of leukemia is the sixth among all kinds of tumors in China.The incidence of childhood leukemia is ranked first in children’s malignant tumors.Although in the past few decades,the treatment of leukemia has made great progress.However,because of the complexity and heterogeneity of leukemia,the treatment of leukemia has always been challenging.This complexity is partly due to the interaction between leukaemia and its microenvironment.In the past ten years,the mechanism of the role of bone marrow microenvironment in the occurrence,development and drug resistance of leukemia is a hot field in the study of hematological malignancies.Bone marrow microenvironment is a structural unit composed of bone marrow stromal cells,adipocytes,mesenchymal stem cells,osteoblasts,osteoclasts,and extracellular matrix,cytokines,osteopontin,calcium ions and so on.Soluble cytokines in the bone marrow microenvironment and extracellular matrix support the growth,proliferation and differentiation of hematopoietic cells.By secreting soluble cytokines,cell adhesion,up-regulating drug resistance genes,regulating leukemia cell metabolism and altering cell cycle,Leukemia cells can evade the killing of chemotherapy drugs.At present,most of the published experimental data are based on 2D cell culture conditions,the conditions are simple and inconsistent with that in vitro,can not fully reflect the real cell physiology.They change the cell’s specific structure(polarity,shape),biochemical signal,cell-cell,cell-matrix signal transduction.Although two-dimensional culture helps us to understand the biological behavior of cells,it has now been demonstrated that 2D culture systems result in different cellular biological activities than in vivo.For example,some important cancer cell characteristics,such as gene mapping or tumor heterogeneity,can not be correctly expressed in two dimensional culture conditions.Animal models are another approach to studying molecular mechanism. owever,many experimental animals have been destroyed by the immune system and can not provide the same substrate-tumor interaction as humans do,preventing the laboratory Research results to the effective conversion of clinical applications.Almost less than 8% of animal models are consistent with clinical trials.Therefore,the switch from 2D to 3D cell culture is a clinical need to better interpret the complexity of tumor cell biology.The 3D cell culture platform overcomes these limitations by maintaining the original shape,cell polarity and genetic character of the tumor and matrix cells.Leukemia is still a killer that threatens the safety of human life,But chemotherapy can not cure leukemia(except for a few types).The prognosis of leukemia has been greatly improved.Despite the improvements in cell morphology,molecular biology,cytogenetics and immunology,Relapse and drug resistance of leukemia are still an international problem.Hematopoietic stem cell transplantation is the only way to cure leukemia.However,the high cost,easy to relapse,severe GVHD side effects and technical limitations make hematopoietic stem cell transplantation hard to carry out for most leukemia patients in China.In the case of relapsed and refractory leukemia,the success rate of hematopoietic stem cell transplantation is low.In recent years,the prognosis of many hematological malignancies has been greatly improved because of the new drugs.Through studying the signaling pathways of leukemia cells and finding the possible pathogenesis of leukemia,developing targeted drugs are the research hotspots in the field of hematological malignancies.For example,Imatinib,dasatinib and borteitin improve prognosis of chronic myeloid leukemia,rituximab makes the efficacy of B-cell non-Hodgkin’s lymphoma significantly improved,Bortezomib,a proteasome inhibitor,significantly prolongs survival and improves quality of life in patients with multiple myeloma.3D cell culture,by mimicking bone marrow microenvironment,reflects the true state of leukemic cells in vivo and provides a reliable tool for the development of targeted drugs.Discoidin domain receptor 1(DDR1),a new receptor tyrosine kinase,is known as the extracellular domain structure is similar to the agglutinin discoid structure.Natural collagen is the ligand of DDR1,and the normal DDR1 signal regulates the basic physiological processes(such as mammary gland development,renal function,etc.).It is found that abnormal DDR1 signals involve multiple kinds of human cancers,and are widely overexpressed in breast cancer,ovarian cancer,brain cancer,lung cancer,myeloid leukemia,pituitary adenoma,glioma,which are related to tumor proliferation,differentiation,invasion and drug resistance.However,the expression and function of DR1 in acute lymphoblastic leukemia cells is not clear.Research purposes: To compare the effects of 2D and 3D culture on the Jurkat cell proliferation,differentiation and expression of DDR1 protein.By blocking DDR1 with inhibitor DDR1-IN-1,we analyzed whether there was any change in sensitivity of Jurkat cells to chemotherapeutic drugs,further analysis of possible signaling pathways provided some guidance for the development of clinical targeted drugs.Methods:1)Using different concentrations of rat tail collagen coated scaffold,the same method was used to seed the same number of Jurkat cells on the scaffold.After 4h,calculate the different concentration of rat tail collagen coated scaffold for effects of Seeding efficiency.The highest Seeding efficiency scaffold was used in subsequent trials.2)The effects of 2D and 3D culture on the proliferation of Jurkat cells were compared by measuring the OD values in 2D and 3D culture system at different time points using CCK-8.3)Flow cytometry was used to determine whether the Jurkat cell phenotype was changed in different culture systems(2D culture system,rat tail collagen coated 3D scaffold culture system,uncoated 3D scaffold culture system),to demonstrate the suitability of 3D scaffolds as a Jurkat cell culture system.4)The proliferation and morphology of Jurkat cells on the 3D scaffold were observed by scanning electron microscopy.5)Western Blot was used to detect the Jurkat cells protein expression of DDR1,STAT3 and p-STAT3 in in different culture systems.6)The sensitivity of Jurkat cells to chemotherapeutic drugs in different culture systems(2D culture system,rat tail collagen coated 3D culture system,uncoated 3D scaffold culture system)was determined by CCK-8.7)Using inhibitor DDR1-1N-1 to block DDR1 protein function,CCK-8 was used again to determine the sensitivity of Jurkat cells to chemotherapeutic drugs in different culture systems(2D culture system,rat tail collagen coated 3D culture system,uncoated 3D scaffold culture system).Results1)Different concentrations of rat tail collagen(0,10,20,40,80,160,320 ug / ml)coated scaffold,by measuring the number of cells in the culture medium indirect response cell seeding efficiency,the results showed that 80ug/ml tail collagen coated scaffold had the highest seeding efficiency.2)The effects of 2D and 3D cultures on cell proliferation were compared by CCK-8 and showed that 3D cells lagged in initial stage compared to 2D culture system.However,with the prolongation of time,the proliferation of 3D cultured cells was obviously better than that of 2D culture,especially after 144 h and 168 h,and the rat tail collagen-coated 3D scaffolds showed superiority to uncoated 3D scaffolds and the cell proliferation could be maintained longer time.3)After cultured for 168 hours in 3D system,the immunophenotypes of cells were detected by flow cytometry and found no significant changes compared with the 2D system.(CD2+CD3+CD4+dim CD8-CD34-CD45+dim)4)Jurkat cells were suspended in media,and a small amount of cell aggregation was found in 2D.Jurkat cells aggregated and adhered to each other in 3D scaffold.With the prolongation of time,cells aggregated into a "sphere" by scanning electron microscopy5)The results of Western Bolt assay showed that the expression levels of DDR1 and p-STAT3 were different.The expression of DDR1 and p-STAT3 was the highest in rat tail collagen-coated 3D scaffold culture system,the lowest in 2D cell culture system.uncoated 3D scaffold culture system was in both between.STAT3 protein expression was no significant difference.6)Jurkat cells in different culture systems show different sensitivities to cytarabine(Ara-c)and daunorubicin(DNR).Jurkat cells revealed high sensitivity to Ara-C cultured on 2D system.Cell proliferation reduced to 89.58% ± 3.26%,62.86% ±6.84% and 53.36% ± 3.37% for 48,144 and 168 hours respectively.Cells cultured on rat tail collagen coated 3D culture system showed resistance to drug treatment,as shown by the result that cell proliferation was 103.98% ± 5.3%,88.69% ± 2.19%,77.11% ± 3.47% at 48,144 and 168 hours.Meanwhile,the drug sensitivity of cells cultured on uncoated 3D scaffold culture system was in both between,with the relative percentage of 87.98% ± 3.74%,73.28% ± 5.5% and 63.65% ± 0.81% at48,144 and 168 hours.A significant death of Jurkat cells was observed in 2D(57.67%± 8.02%,42% ± 4.58%)when compared to rat tail collagen coated 3D culture system(78% ± 5.57%,57.33% ± 5.03%)in the presence of DNR for 96 and 168 hours.A similar trend of combination use of Ara-C and DNR was obtained in 2D(24.67% ±4.73%)and uncoated 3D scaffold culture system(39% ± 7.55%)in comparison to rat tail collagen coated 3D culture system(46.33% ± 4.04%)for 168 hours.7)Using inhibitor DDR1-1N-1 to block DDR1 protein function,the chemosensitivity of urkat cells in different culture systems to cytarabine and daunorubicin were significantly increased.Jurkat cells were substantially completely wiped out after 144 h.Conclusions1)Scaffolds coated with different concentrations of rat tail collagen could increase the adhesion of Jurkat cells to the scaffold,and 80ug/ml tail collagen coated scaffolds are more conducive to cell adhesion and proliferation.2)3D scaffold could better mimic the bone marrow microenvironment,cell growth closer to that in vivo,more conducive to the long-term survival of cells.3)The expression level of DDR1 and p-STAT3 protein in rat tail collagen modified 3D scaffold culture system increased to varying degrees,suggesting that collagen as DDR1 ligand can stimulate DDR1 expression.4)Polycaprolactone 3D culture system does not change the cellular phenotype,does not cause further differentiation.5)The sensitivity of Jurkat cells to chemotherapeutic drugs is different in different culture systems.The cell sensitivity to chemotherapeutic drugs in 3D culture conditions was lower than that in 2D culture conditions.6)Using inhibitor DDR1-1N-1 to block DDR1 protein function,the chemosensitivity of Jurkat cells increased,suggesting that DDR1 may be involved in Jurkat cell resistance mechanism.Inhibiting or antagonizing the function of DDR1 maybe increase the sensitivity to chemotherapeutic drugs. |
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