| BackgroudLeukemia is a malignant hematopoietic disease, which is the highest incidence rates and also is one of the main cause of death of children. More than 90% of leukemia in children are acute leukemia, and acute lymphoblastic leukemia accounts for about 2 / 3. In the past 20 years, children with acute leukemia has made great progress, especially in ALL, which 5 years event free survival(EFS) reached 70% ~ 80%.Previously, the most effective treatment of the disease is chemotherapy. But recurrence will take place finally in part patients after they underwent initial effective chemotherapy, the most important reason is chemotherapeutic drug resistance produced by the leukemia cells. The most mechanism of drug resistance among tumor cells is that the apoptosis induced by chemotherapeutic drug is blocked. So, the hindrance to effective leukemia treatment is drug resistance and becomes a problem in leukemia treatment domain.People focus their attention on hematopoietic microenvironment (HM) much more because HM plays an important role in leukemia drug resistance. In the past decade, the studies on HM have got much more conclusions and found that the way which HM induces leukemia drug resistance includes two sides. One is that the cell adhesion molecule-drug resistance (CAM-DR) occurred in tumor cells and the other is that soluble cytokine molecule-drug resistance (SM-DR) induced drug resistance and decrease of apoptosis.HM is composed of bone marrow stromal cells (BMSC),extracellular matrix (ECM),cytokine and mesenchymal stem cells (MSCs).MSCs are the predecessor of BMSC, which are the stem cells besides hematopoietic stem cells (HSCs) in bone marrow. The cell surface immune phenotypes of MSCs expressed CD29,CD44,CD73,CD105,CD166.In special culture, MSCs had potential to cell differentitation in vitro, and could produce many cytokine regulating hemotopoiesis and growth, such as IL-6,IL-7,IL-8,IL-12,M-CSF.MSCs play an important role on regulating HM. Although MSCs were few in HM, the study on it found that it was important to hemotopoiesis.Normal MSCs play key on the process of hemotopoiesis in organism. They not only affected the proliferation, differentitation and survival of HSCs by secreting cytokine cell adhesion molecule (CAM) but also adhere directly. More and more studies found that MSCs took part in the occurrence, development of leukemia and closely related to the drug resistance and apoptosis resistance. MSCs is one of the key factor affecting the proliferation, differentitation and survival of the B lymphocyte HSCs and B lymphocyte leukemia. Study on myelocytic leukemia indicated that MSCs could decrease the sensitivity to chemotherapeutic drug of leukemia cells, because MSCs could block the escape from chemotherapy. But most people focused attention on normal MSCs, MSCs of lymphoblastic leukemia children haven't been studied.It was confirmed that the effect on inhibitory apoptosis of drug induced leukemia was of HM. But it was complicated to explain the effects on each other and the roles of components.In ALL children,the mechanisms of ALL children MSCs and leukemia cells had effect on each other and induced drug resistance hasn't understood. So, it will promote the study in depth if we find a new ideology and could help us explain the mechanism of MSCs protect leukemia cell and escape apoptosis.Objective1. Study the method of MSCs separation and proliferation in vitro of ALL children.2. Study the effect of MSCs on leukemia cell strains,K562,K562/AO2 and explain the characteristic of MSCs regulating leukemia proliferation. 3. Analysis the effect of MSCs on chemotherapeutic sensitivity of leukemia and explore it's regulating mechanism and provide experiments basis to leukemia drug resistance and establish orientation of research.Content1. We established the method that MSCs of ALL children separation and proliferation in vitro.2. Leukemia cell strains, K562 and K562/AO2 adhesioned to MSCs and co-cultured to MSCs and study the effect on proliferation of leukemia cells.3. The research to observe chemotherapeutic sensitivity of leukemia cells adhesion to MSCs.4. Research the mechanism of drug resistance of MSCs induced leukemia cells.Line of technique Method1. Gain of MSCs from ALL children bone marrow by divided, cultured and identification.2. Groups: the co-culture of MSCs and K562;K562 suspension alone; the co-culture of MSCs and K562/AO2;K562/AO2 suspension alone.3. After six days cultured,measuring leukemia cells growth curve.4. Measuring cell cycle after 72-hour co-culture by flow cytometry.5. Four groups treated with different concentrations of ADM and measured apoptosis by flow cytometry.6. Established real-time PCR to detect mdr1,Bcl-2 and Bax gene.Result1. We established the method of MSCs'separation and proliferation was viable and steady.2. Compared with the cell growth curve of leukemia cells alone, the leukemia cells co-cultured with MSCs grew slower and the exponential phase of growth was not significant.3. The results of measuring cell cycle after 72-hour co-culture by flow cytometry showed that compared with the suspension culture group, the leukemia cell cycle progression was blocked in adhesion group. Leukemia cells had an increase in the percentage of cells in G0-G1 phase (P<0.05),decreased in G2-M phase (P<0.05).4. In this work, leukemia cells in suspension culture group and adhesion culture group at different concentrations of ADM and apoptosis rates were observed after 24h.The outcome showed that the suspension culture group's apoptosis rates was obviously more than adhesion culture group.5. In our study,mdr1 gene expression in the mechanism of the classical multidrug resistance (MDR) of K562 and K562/AO2 cells after adhesion culture was detected by using the real-time PCR technology. It showed that adhesion culture did not only induce mdr1 expression in K562 cell but also expression more in K562/AO2 cells.Bcl-2 gene of adhesion culture group which we detected also by RT-PCR expressed higher than suspension culture groups. Bax gene of adhesion culture group which we detected expressed similar to suspension culture groups.Conclusion1. ALL children MSCs suppressed the growth of K562 and K562/AO2 cells in vitro. Adhesion made leukemia cells depress sensitivity to ADM.2. Adhesion to MSCs made leukemia cells cell cycle blocked and most cells blocked in G0-G1 phase.3. The result of PCR suggested that drug-resistant was related to Bcl-2 gene and has nothing with mdr1 gene.
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