Different Effects And Mechanism On K562Cells Growth Regulation By Two Kinds Of Bone Marrow Mesenchymal Stem Cells

Objective:Just like the solid tumor, the neoplastic hematologic disorder is alsorelated to the activation of proto-oncogene and (or) inactivation of cancersuppressor gene with respect to its genesis and development. However, theneoplastic hematologic disorder is different from the solid tumor in itsgrowing and proliferating environment, for it is developed in a fine yetcomplicated regulation system—hematopoietic microenvironment. In normalsituation, the stromal cells of regulating activity in the hematopoieticmicroenvironment regulates the growth of hemocytes and maintains thenormal forms of hemocytes in different stages through the formation andrelease of the hematopoietic factors and hematopoietic inhibiting factors.As a kind of malignant clone disease originating from multifunctionalhematopoietic stem cells. The chronic myelocytic leukemia (CML) is usuallycharacterized by the abnormal increase of peripheral blood leucocytes whilethe CML leukemia cells characterized by multiplied neutral myelocytes,metamylocytes, mature granulocytes, eosinophilic granulocytes, andbasophilic granulocytes. Compared with normal myelogram and hemogram,CML displaying the aforesaid changes has interrupted the normal balance ofhematopoiesis regulation with the reproduction and differentiation of thetumor cells. The changes also remind us about the abnormal change in thehematopoietic microenvironment of CML patients’ bone marrow.In order to determine the effect of marrow’s hematopoietic micro-environment on the proliferation of CML’s tumor cells, we adopted bonemarrow derived mesenchymal stem cells (BMMSCs) to carry out in vitrosimulation of the marrow gene cells in hematopoietic microenvironment, forBMMSCs were stem cells of stromal cells of hematopoietic microenvironment that could be differentiated to be all sorts of stem cells and secrete severalkinds of cell factors to take part in the regulation of bone marrow’shematopoietic function. In accordance with our previous studies, such genes inToll receptor pathway as TAB1, TBK1, MAPK11, CXCL10displaysignificant difference in the expression within leukemia cells of CMLchromatic and acute transformation phases. We attempted to find out whetherthe difference played a role in regulating the generation and proliferation ofCML leukemia cells.Human mesenchymal stem cells (HMSC) of bone marrow were collectedfrom both healthy people and patients suffering from CML in the chromaticphase so as to make in vitro simulation of the bone marrowmicroenvironments of healthy people and CML patients. K562cell lines wereused to simulate CML leukemia cells and they were co-cultured with themesenchymal stem cells of bone marrow to construct a model similar to thehuman’s marrow cavity in which the hematopoietic microenvironment exertedregulation on the leukemia cells of CML. Growth curves of K562cells weredrafted, the cell factors including IL-6, IL-8, TNF-α, and IFN-α in thesupernatant of co-cultured cells were detected, genes TAB-1, TBK1andNF-κB and protein expressions of bone marrow mesenchymal stem cells aswell as the gene β-catenin and protein expressions in K562cells cultured indifferent ways were compared in order to study the different effects of bonemarrow mesenchymal stem cells on the growth of K562cells and putpreliminary study about the possible mechanism of Toll receptor pathway inthe regulating process.Methods:1Part one：(1) Groups of Experiments, the co-culture of K562cells with bone marrowmesenchymal stem cells of patients suffering from chromatic myeloidleukemia (K562+CML-BMMSCs) and the co-culture of K562with the bonemarrow mesenchymal stem cells of healthy people (K562+HMSC-hm) weredivided to be Experimental Group1and2, respectively, meanwhile a group of K562in the way of independent culture was set as the Control Group.(2) CCK8was adopted to draft the growth curve of K562.(3) Apoptosis of K562cell detected by flow cytometry.2Part two：(1) Groups of Experiments：the co-culture of CML-BMMSCs with K562cellsand the co-culture of HMSC-hm with K562cells were divided to beExperimental Group1and2, respectively, meanwhile the independent cultureof CML-BMMSCs and HMSC-hm were set as Control Group1and2,respectively.(2) RT-PCR was employed to detect the expression of gene TAB1, NF-κB andTBK1in the four groups of BMMSCs.(3) Western blot was employed to detect the protein products of TAB-1,NF-κB and TBK1in the four groups of BMMSCs.(4) The cell factor secretion levels of IL-6, IL-8, TNF-α, and IFN-αin the fourgroups were detected and compared with enzyme linked immunosorbent assay(ELISA).3Part three：(1) Groups of Experiments：theco-culture of K562cells with CML-BMMSCsand HMSC-hm were divided to be Experimental Group1and2, respectively,meanwhile the independent culture of K562cells was set as Control Group1,K562cells affected by interferon-α2b for72h was set as Control Group2.(2) RT-PCR was employed to detect the expression of geneβ-catenin in thefour groups of K562cells.(3) Western blot was employed to detect the protein ofβ-catenin in the fourgroups of K562cells.Results:1The K562cells growth curves of three groups revealed a relatively slowgrowing speed of K562cells in three groups during0-48h, but an acceleratedone during48-72h. Compared with other two groups, K562cells inExperimental Group2were a bit slower in growing which meant itsproliferation was inhibited to some degree, the different was significant (P<0.05). However there was no significant difference in the proliferationspeed of K562cells in Experimental Group1and Control Group (P>0.05).2As three groups of K562cells cultured72h measured by flow cytometry, theresults observed in early and late stage apoptosis cells was1.52±0.10forExperimental Group1,13.33±7.68for Experimental Group2, and2.46±0.88for Control Group. The Experimental Group2had the highest value that wassignificantly different from that of Experimental Group1and Control Group(P<0.05). The Experimental Group1and Control Group were notsignificantly different from each other (P>0.05).3Testing results of bone marrow mesenchymal stem cells of four groups:NF-κB, TAB1and TBK1through RT-PCR(1) The NF-κB/β-actin mRNA expression value was0.0402±0.0024forExperimental Group1,0.0196±0.0025for Experimental Group2,0.0214±0.0028for Control Group1, and0.0188±0.0036for Control Group2.Among four groups, the NF-Κb/β-actin OD value of Experimental Group1was the highest, significantly different from Experimental Group2, ControlGroup1and2(P<0.05). The value of Experimental Group2was significantlydifferent when compared with that of both Control Group1and2(P>0.05).The two control groups were not significantly different from each other(P>0.05).(2) The TAB1/β-actin mRNA expression value was0.0319±0.0086forExperimental Group1,0.0224±0.0051for Experimental Group2,0.0252±0.0030for Control Group1, and0.0134±0.0046for Control Group2. Amongthose groups, Experimental Group1had the highest value that wassignificantly different from that of Control Group2(P<0.05), but not from thatof Experimental Group2and Control Group1(P>0.05). There was significantdifferent between the values of Experimental Group2and Control Group2(P<0.05), but not between Experimental Group2and Control Group1(P>0.05). Compared with Control Group1, Control Group2was sig-nificantly lower (P<0.05).(3) The TBK1/β-actin mRNA expression value was0.0406±0.0080for Experimental Group1,0.0584±0.0094for Experimental Group2,0.0504±0.0091for Control Group1, and0.0614±0.0130for Control Group2. Acomparison showed the value of Control Group2to be the highest one whichwas significantly different from that of Experimental Group1(P<0.05), butnot from that of Experimental Group2and Control Group2(P>0.05). Therewas no significant difference among the values of Experimental Group1,2and Control Group2(P>0.05).4Testing results of NF-κB, TAB1and TBK1protein products in bone marrowmesenchymal stem cells of four groups with Western blot(1) The all four groups of bone marrow mesenchymal stem cells couldexpress NF-κB protein, but the protein band of Control Group2wasapparently restricted when compared with other three groups. The imageanalysis software—Quantity One was used to analyze the optical density valueof NF-κB protein band and produce corresponding bar chart. According to theresults, the NF-κB protein concentration order going from high to low was:Experimental Group1, Control Group1, Experimental Group2and ControlGroup2.(2) The all four groups could express TAB1protein, but the protein band ofControl Group2was apparently restricted when compared with other threegroups. The analysis of the TAB1protein band’s optical density value with theimage analysis software—Quantity One showed the protein concentrationorder in four groups going from high to low was: Experimental Group1,Control Group1, Experimental Group2and Control Group2.(3) The all four groups could express TBK1protein. However the proteinbands of Experimental Group1and Control Group1were relatively narrow,while that of Experimental Group2and Control Group2was wider. Theanalysis of TAB1protein band’s optical density value with Quantity Onerevealed the TBK1protein concentration order going from high to low amongfour groups was: Experimental Group2, Control Group2, Control Group1and Experimental Group1.5Detecting results of IL-6, IL-8, TNF-α and IFN-α in the supernatant of cells in four groups with ELISA(1) The OD450value of IL-6was23.75±1.32for experimental group1,25.21±0.30for experimental group2,16.47±0.94for control group1,14.62±0.92for control group2. The value of experimental group2was thehighest and it was significantly different (P<0.05) from the values of theseparately the two control groups, but not from the experimental group1(P>0.05). There was also significant difference with respect to OD450valuebetween experimental group1and the two control groups (P<0.05), as well asseparately cultured control group1and control group2(P<0.05).(2) The OD450value of IL-8was1269.28±4.12for experimental group1,6022.64±134.71for experimental group2,66.26±11.56for control group1,and127.26±4.49for control group2.. The experimental group2displayed thehighest value significantly different from that of other three groups (P<0.01).The values of the experimental group1was also significantly different fromthe two control groups (P<0.01). Nevertheless, there was no significantdifference between the two control groups (P>0.05).(3) The OD450value of TNF-α was32.17±0.83for experimental group1,36.33±7.03for experimental group2, and50.79±4.01for control group1and44.17±3.09for control group2. The value of control group1was the highest,significantly different from the two experimental groups (P<0.05), but notfrom control group2(P>0.05). Compared with experimental group2, thevalue of control group2was significant higher (P<0.05). There was nosignificant difference between the two experi-mental groups (P>0.05).(4) The OD450value of IFN-α was27.73±2.83for experimental group1,40.11±0.61for experimental group2,37.93±1.86for control group1, and53.66±4.29for control group2. The control group2had the highest valuewhich is significantly different from the values of other three groups (P<0.05).The values of experimental group2was significantly different fromexperimental group1(P<0.05), but not control group1(P>0.05). Comparedwith control group1, the values of experimental group1was significantlylower (P<0.05). 6The β-catenin/β-actin mRNA expression value of K562cells in four groupswas0.0130±0.0025for Experimental Group1,0.0097±0.0020for Exper-imental Group2,0.0265±0.0042for Control Group1, and0.0096±0.0027forControl Group2. Among the four groups, Control Group1revealed thehighest value which was significantly different from that of ExperimentalGroup1,2and Control Group2(P<0.05). We can’t see any significantdifference among the results of Experimental Group1,2and Control Group2(P>0.05).7The Western blot detection result of β-catenin revealed the bone marrowmesenchymal stem cells of all four groups could express the β-catenin protein.In accordance with the analyzing result about the optical density value ofβ-catenin with image analysis software—Quantity One, the β-catenin proteinconcentration order going from high to low among four groups was ControlGroup1, Experimental Group1, Experimental Group2, and Control Group2.Conclusions:1The human bone marrow mesenchymal stem cell strains from healthpersons could restrain the growing of K562cells, and the human bone marrowmesenchymal stem strains from patients with chronic myelogenous leukemiacould not inhibit the growing of K562cells.2The different influences to K562cells of the two kinds of two bonemarrow mesenchymal stem cells may be realized through modulating theactivity of Toll sample receptor pathway. The human bone marrowmesenchymal stem cell strains could release the cell factors like IL-6and IL-8through the inhibition of the classic MyD88dependence channel, and promotethe release of the modulates of cell factors like TNF-α and IFN-α throughnon-MyD88dependence channel, which would realize the inhibition for thegrowing of K562cells. On the contrary, the classic activated MyD88dependence channel of the human bone marrow mesenchymal stem strainsfrom patients with chronic myelogenous leukemia would release large amountof factors like IL-6and IL-8that could promote the growing of cells.Meanwhile, it would also reduce the activity of the non-My-D88dependence pathway and lower the secretion of cell factors that would restraining thegrowing of K562cells like TNF-α and IFN-α.3Both two bone marrow mesenchymal stem cells could down-regulatethe activity of the Wnt/β-catenin for K562cells.