| BackgroundLeukemia stem cells is the source of recurrence of leukemia after chemotherapy, there is evidence that leukemia stem cells settled in the hematopoietic niche. The so-called hematopoietic niche is composed of three-dimensional hematopoietic microenvironment by bone marrow stromal cells, osteoblasts, and endothelial cells. According to the study found that the hematopoietic niche through a variety of mechanisms of leukemia stem cells remain in the cell cycle Go phase to evade the killing effects of conventional chemotherapy drugs. Leukemic stem cells to various mechanisms for the protection of the hematopoietic niche, it must be stable anchor in the micro environment, adhesion molecules and adhesion molecule receptors is to leukemic stem cells in the hematopoietic niche acting factors. Therefore, by reducing adhesion molecule expression of leukemia stem cells may prompt more leukemic stem cells from the hematopoietic niche, enter the proliferation of the division cycle, improve the effect of the chemotherapy drugs, which may become a new way to reduce the recurrence of leukemia after chemotherapy. Cell surface of hematopoietic stem cells have been found more than20kinds of adhesion molecules, the structural characteristics of these adhesion molecules can be divided into the following five categories:(1) integrin family;(2) of the immunoglobulin superfamily;(3) prime family;(4) mucin-like vascular addressin;(5) cadherin or calcium dependent cell adhesion molecule family; In addition, there are some yet to be classified adhesion molecules, such as of CD44in The VLA-4(of CD49d) is one of the integrin family, interaction with VCAM-1mediated cell adhesion; the VLA-4(of CD49d)-mediated residual leukemia and drug-resistant adhesion molecule, using anti-the VLA-4(of CD49d) monoclonal antibody in combination with chemotherapy can eliminate residual leukemia in the mouse model. CD44belongs to the unclassified adhesion molecules, the main ligand is hyaluronic acid, and of CD44by receptor binding of leukemia cells anchored in the bone marrow microenvironment conducive to the value-added of the leukemia cells and prevent differentiation. Targeted CD44monoclonal antibody will enable the leukemic stem cells tend to differentiate, which. also proved by blocking the adhesion between leukemia cells and hematopoietic niche can overcome the differentiation blocked induction of cell maturation. Vermeulen and found antibodies block the inhibition of adhesion molecules of the VLA-4(of CD49d) and CD44on murine hematopoietic stem cells into human bone marrow up to77%～86%. Therefore, we chose of CD49d and CD44, as observed indicators. The mechanism of arsenic trioxide leukemia induced differentiation, apoptosis, and cytotoxicity, little research on its leukemic stem cell adhesion molecules and the role of the hematopoietic microenvironment. KG1a cells derived from acute myeloid leukemia cell lines, poorly differentiated cell phenotype of CD34+of CD38-reactive colony-stimulating factor, hematopoietic stem/progenitor cell lines, but also in vitro studies the leukemia cells function features a useful model. This study analog of bone marrow stromal cells in vitro hematopoietic microenvironment, and KGla cells co-cultured to observe the impact of arsenic trioxide on KG1a cell surface adhesion molecule CD44, of CD49d expression rate.Methods1. Bone marrow stromal cells culture:After informed consent, fresh bone marrow from normal or leukemia patients were collected, centrifuged to cultivate, until the cells cover the80%of bottom, with0.25%trypsin digested cells and passaged.2. Bone marrow stromal cells from normal or leukemia patients co-cultured with KGla cells to found hematopoietic microenvironment in vitro:Take better activity, logarithmic phase of bone marrow stromal cells, cultured in24-well plates for24h, the PBS wash away adherent cells; KG1a cells were inoculated on the bone marrow stromal cell layers, cultured for24h.3. MTT assays for the determination of arsenic trioxide on the half inhibitory concentration (IC50) of KGla cells:MTT assays were performed to determine inhibition of cell proliferation rate of arsenic trioxide. Inhibition of cell proliferation rate (%)=[1-(experimental group OD-blank group OD)/(control group OD-blank group OD)]×100%. Probit analysis with SPSS software program to calculate the IC50values of24hours of arsenic trioxide, and to determine the experimental drug concentration.4. Detected the expression of the surface adhesion molecules CD44and CD49d of KGla cell by Flowcytometry:KGla cells in the different condition groups to collected, conjugated with PE-anti-human of CD49d and FITC-anti-human of CD44, set the isoform control group. Incubate for30minutes away from light, collected10000cells to detect the expression rate of CD44and CD49d by flowcytometry.5. Statistical analysis:The above experiment was repeated three times. The statistical data are expressed as X±S. Using SPSS17.0statistical software to analyze, Probit analysis to calculate the IC50values; The regression equation with the goodness of fit test; One-way ANOVA analysis was used to analyze difference in different experiment groups. LSD test was used in intergroup compared. Observed differences were regarded as significant if P<0.05.Results1. Bone marrow stromal cells morphological observation:Cultured to10or12days, there were many colony in the bottom of culture bottle, after15or20days, single-celled layer formed fusion growth. After passaged generation, colony formed fast, cell morphology were homogeneous. After KGla cells and stromal cells cultured for24hours, there were slabstone form of proliferation.2. MTT assays to determine the arsenic trioxide KGla cells to half of the concentration of suppression (IC50):With the increase of the concentration, arsenic trioxide on KG1a cell growth has certain inhibition (F=2680.604, P=0.000). With SPSS analysis software probit analysis program, calculated IC50value of arsenic trioxide KG1a cells of24hours. IC50was4.654u mol/L,95%confidence interal (3.634,5.715)μmol/L,(goodness-of-fit testing X2=1.840, P=0.399). To avoid the apoptosis cells too much, too fragmented and cause streaming specimen affect the results, the experimental intends to use less than1/2IC50value concentration for the experimental drug concentration (final concentration for0.25u mol/L,0.50u mol/L,1.00u mol/L,2.00u mol/L).3. Detected the expression of the surface adhesion molecules CD44and CD49d of KGla cell by Flowcytometry:3.1Effect on the expression of cell surface adhesive molecular of KG1a cells by bone marrow stromal cells:The expression rate of the surface adhesion molecules CD44and CD49d of KG1a cells in three groups were93.50±0.85%and 42.68±0.78%;98.31±0.50%and51.49±1.55%;99.13±0.43%and58.15±0.67%. The difference was statistically significant (F=71.487, P=0.000; F=155.98, P=0.000). Between groups, the expression rate of KG1a cell surface adhesion molecules CD44and CD49d in group B and group C were significantly higher than group A (P=0.000). In group C, KG1a cell surface adhesion molecules CD49d also significantly higher than group B (P=0.000). But between the group B and group C, the expression rate of the surface adhesion molecules CD44of KG1a cells was no significant difference (P=0.159). Respectively with leukemia bone marrow stromal cells cultured24hours,36hours,48hours later, the expression rate of KGla cell surface adhesion molecules CD49d and CD44was99.04±0.27%and58.32±1.07%;99.03±0.29%and57.92±0.80%;99.40±0.19%and59.52±1.72%. The difference between different time points was no statistically significant (F=2.139, P=0.199; F=1.318, P=0.335).3.2Effect of Different concentration arsenic trioxide on the expression of CD44and CD49d of BMSCs co-cultured KG1a cells:With different concentration of arsenic trioxide (0.25u mol/L,0.50u mol/L,1.00u mol/L,2.00u mol/L), when the drug concentration increased to2.00umol/L, The expression rate of surface adhesion molecules CD44and CD49d of KGla cells decreased from99.22±0.21%and59.27±0.80%to85.25±0.50%and35.75±3.94%, with significant difference (F=117.231, P=0.000; F=30.536, P=0.000). Between groups, the express rate of CD44have statistically significant (P<0.05); the expression rate of CD49d between0.25μmol/L and0.50μmol/L group (P=0.066) and0.50μmol/L and1.00μmol/L group (P=0.106) was no statistically significant difference.3.3Effect on the expression of CD44and CD49d of BMSCs co-cultured KGla cells in different length of time by1μmol/L arsenic trioxide:With1.00u mol/L arsenic trioxide for respectively24hours,36hours,48hours, the expression rate of KG1a cell surface adhesion molecules CD44and CD49d were94.32±0.77%and46.14±0.81%;92.28±0.62%and41.66±1.07%;87.89±1.45%and34.40±1.62%. the difference was statistically significant (F=31.494, P=0.001; F=71.054, P=0.000). Comparative between groups were statistically significant difference (P<0.05).Conclusion(1) Arsenic trioxide can inhibit the growth of KGla cells, the IC50value of Arsenic trioxide for24h is4.654μmol/L,95%confidence interval (3.634,5.715) μmol/L,.(2) Bone marrow stromal cells can upregulate the expression of surface adhesion molecules CD44and CD49d of KG1a cells, this effect does not increased in different length of time; leukemia bone marrow stromal cells can upregulate the expression of CD49d significantly than the normal bone marrow stromal cells.(3) In vitro, arsenic trioxide can be significantly downregulate the expression of surface adhesion molecules CD44and CD49d of KGla cells with time-dose dependent manner. |
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