The Study Of Effects Of Hematopoietic Microenvironment From Human Umbilical Cord Blood-derived Stromal Cells On The Residual Leukemia Cells And Its Mechanisms

Minimal residual disease (MRD) is the residual leukemia cells in vivo after complete remission of leukemia, and the cells are mostly in the G0 phase and not sensitive to chemotherapeutic drugs, which is the major source of refractoriness and relapse for leukemia. At present, we have not yet found a exact leukemic cell-specific antigen. Although we have applied a number of ways collaborated but the kill and remove effect of the MRD can not be satisfied and effective, and the application of chemotherapy drugs and a stronger collaborated chemotherapy brings serious damage to the normal hematopoietic function. For the removal of MRD, we are still lack of an effective and low toxicity method, which has become one of the serious key obstacles for the breakthrough of the treatment of leukemia. To explore the effect and mechanism of hemopoietic microenvironment built by matrix cells from different sources to the MRD has an important theoretical and practical significance for further reducing leukemia relapse and improving long-term survival rate of leukemia.Hematopoietic inductive microenvironment(HIM),which was mainly composed of bone marrow stromal cells(BMSCs), acted as the soil in regulating the growth and development of hematopoietic stem/progenitor cell.The development and proliferation of leukemic cells were dependent on the abnormal HIM.The BMSCs derived from leukemia that supported and protected leukemic cells involved in the occurrence and refractory of leukemia. One of the important mechanisms for the supply of MRD is that BMSCs can help leukemia cells to evade the chemotherapy drugs, so that to explore an effective way to clear MRD starting from the hematopoietic microenvironment is a new entry point.The group's past research has shown that: under the condition of the appropriate combination of cytokines, human umbilical cord blood-derived stromal cells (hUCBDSCs ) can be successfully cultivated and amplified; after the amplification, the cells composition and immunophenotype of the hUCBDSCs is similar to that of hBMSCs, which can secrete a variety of cytokines and carries the similar material basis for supporting and reconstructing the hematopoietic function with hBMSCs, so that it may be called hematopoietic inductive microenvironment from human umbilical cord blood-derived stromal cells (hUCBDSC-HIM).The co-culture of human acute T Lymphocytic leukemia cell line Jurkat cells respectively with hUCBDSC-HIM and hBMSC-HIM has found: hUCBDSC-HIM has a stronger inhibition and the phenomenon of apoptosis promotion to the Jurkat cells compared with hBMSC-HIM.Based on this point, the experiment will study the influences of hemopoietic microenvironment simulated by stromal cells from different sources to the biological characteristics of residual drug resistant Jurkat cells, such as generation cycle disposition, proliferation, differentiation, apoptosis and drug sensitivity systematically and in-depth, and carry on the preliminary study of its mechanisms.Contents and methods:1. Influence of microenvironment of normal and acute lymphoblastic leukemia bone marrow stromal cells to Jurkat cells'biological characteristics respectivelyGroups: Jurkat suspended culture group; Jurkat/nomal hBMSCs co-culture group; Jurkat leukemic hBMSCs co-culture group.The spatial relationship between the Jurkat cells and stromal cells was observed under the inverted microscope and scanning electron microscope. MTT method for the detection of the adhesion rate of Jurkat cell; Transwell method for the detection of Jurkat cell migration and invasion function changes; CCK-8 method, Real-time Q-PCR method, Western blot method for the detection of Jurkat cell proliferation characteristics change; flow cytometry for the detection of Jurkat cell cycle and DNR dose perturbation changes; flow cytometry method, transmission electron microscopy, Real-time Q-PCR method, Western blot method for the detection of Jurkat cells apoptosis; NBT reduction method for the detection of Jurkat cell differentiation level changes.2. Preliminary study on the culture and its drug resistance mechanism of residual drug resistance Jurkat cellWe use the isolated culture of BMSCs in patients with primary acute lymphoblastic leukemia in vitro to simulate the bone marrow hematopoietic microenvironment. In the long process of co-culture with Jurkat cells, we choose the culture medium containing 50ng/ml DNR. With the culture time extended, the vast majority of Jurkat cells are floating dead. A very small number of residual Jurkat cells recover and got the drug resistant ability that can survive and proliferate in the culture medium with 50ng/ml final concentration of DNR, these cells are recorded as Jurkat / DNR cells. We use CCK-8 method to detect the proliferation characteristics and the resistance coefficient of a wide range of chemotherapy drug of Jurkat and Jurkat / DNR cell; flow cytometry method for the detection of generation cycle and the dose perturbation of the DNR for Jurkat and Jurkat / DNR cell; through the detection of Caspase - Glo 3 / 7 activity, Real-time Q-PCR method, Western blot method for the detection of apoptosis-related indicators of Jurkat and Jurkat / DNR cell; Real-time Q-PCR method for multidrug resistance-associated gene of Jurkat and Jurkat / DNR cell : expression of MDR1 and MRP mRNA.3. The influences of hUCBDSCs to the residual drug resistant Jurkat cellsGroups: Jurkat / DNR cell suspension culture group; Jurkat / DNR cells / normal hBMSCs co-culture group; Jurkat / DNR cells / hUCBDSCs co-culture group; Jurkat / DNR cells / leukemia hBMSCs co-culture group (set only sensitive to IDA). Collect the Jurkat / DNR cells after suspension culture and co-cultured 10 days to detect their changes in proliferation, generation cycle disposition, apoptosis, differentiation, drug sensitivity of the IDA and the expression level of multidrug resistance gene MRD1 mRNA.Results:1. Inverted microscope and scanning electron microscopy: With the culture time extended, the normal hBMSCs and leukemia hBMSCs adhere to, combine with, and wrap the Jurkat cells, and they have the shielding function to the co-cultured Jurkat cells, and they will affect its biological characteristics in a certain extent.1.1 Adhesion and migration function: the adhesion and chemotaxis function to Jurkat cells of leukemia hBMSCs is stronger than normal hBMSCs.1.2 Inhibition of proliferation: Inhibition of of Jurkat cell's proliferation by normal hBMSCs is stronger than leukemia hBMSCs.1.3 Generation cycle disposition: compared with the Jurkat cells in suspension culture, the Jurkat cells proportion in G0/G1 + G2 / M phase of the hBMSCs leukemia cells co-cultured group has significantly increased; the proportion of cells in S + G2 / M phase in the normal hBMSCs co-culture group has significantly increased.1.4 For the Jurkat cell apoptosis: hBMSCs leukemia inhibit Jurkat cell apoptosis, while normal hBMSCs promote Jurkat cell apoptosis.1.5 For the Jurkat cell differentiation: the promote-differentiation function of hBMSCs leukemia to the Jurkat cells co-cultured is weaker than normal hBMSCs.1.6 For the DNR dose changes: the dose of Jurkat cells in the co-cultured prostate group was significantly lower than the suspension culture group, of which Jurkat cells in the hBMSCs leukemia co-culture group is the lowest.2. Jurkat cells peg-and-socket joined in matrix cell gradually get the drug resistance continuously stimulated by the DNR, left the DNR resistance of Jurkat cells. Compared with Jurkat cells, Jurkat / DNR cells has the following characteristics:2.1 Proliferation curve: the proliferation speed of Jurkat / DNR cells significantly slow down than Jurkat cells.2.2 Generation cycle disposition: the proportion of cells in G0/G1 phase increased; the proportion of different diploid cells increased.2.3 Apoptosis rate: the occurrence of spontaneous apoptosis of cells significantly reduced.2.4 DNR dosage: the average drug concentration in a single Jurkat / DNR cell markedly reduced.2.5 Multi-drug resistance to a wide range of chemotherapy drug.2.6 Multi-drug resistance-associated gene expression: in the Jurkat / DNR cells, we can detect the expression of MDR1 mRNA; its expression level of MRP mRNA increased.3. hUCBDSCs to the residual drug resistanct Jurkat cells3.1 Proliferation curve: inhibition of cell proliferation of hUCBDSCs to Jurkat / DNR cells is stronger than normal hBMSCs.3.2 Generation cycle disposition: hBMSCs and normal hUCBDSCs can both promote Jurkat / DNR cells to the generation cycle, the proportion of cells in G0/G1 phase of hUCBDSCs co-culture group is the lowest; the proportion of different diploid cells in Jurkat / DNR cells in the co-culture group significantly reduce. 3.3 To the cell apoptosis and differentiation: the promotion of hUCBDSCs to the spontaneous apoptosis and differentiation of co-cultured Jurkat / DNR cell is stronger than normal hBMSCs.3.4 Sensitivity to IDA: hUCBDSCs and hBMSCs can increase the IDA sensitivity of Jurkat / DNR, and hUCBDSCs is superior to hBMSCs.3.5 mRNA expression in MDR1: the expression of MDR1 mRNA level in co-culture group of Jurkat / DNR cell is lower than the suspension culture group, of which the expression level of the hUCBDSCs co-culture group is the lowest.Conclusion:1. Leukemia bone marrow stromal cells micro-environment can contribute to the shelter, growth and evasion of the anti-drug chemotherapy of leukemic cell; compared with Leukemia bone marrow stromal cells, normal bone marrow stromal cells has a stronger function in pro-apoptotic, differentiation and inhibit proliferation to Jurkat cells . At the same time, it can increase the sensitivity of the DNR of Jurkat cells.2. The hematopoietic micro-environment simulated by leukemia bone marrow stromal cells can induce Jurakt cells to form multi-drug resistance, which is an important reason for the formation of residual drug resistance, suggesting that it is possible to inhibit the formation of residual leukemia and reduced recurrence by transforming the hematopoietic microenvironment in patients with leukemia.3. Bone marrow-derived stromal cells and cord blood-derived stromal cells can both transform the hematopoietic microenvironment of leukemia, and reverse the drug resistance of Jurkat / DNR cells in a certain extent.4. Cord blood-derived stromal cells have a stronger effect in the transformation of hematopoietic microenvironment of leukemia and the reverse of Jurkat / DNR cell drug resistance.