| Some experimental studies of the bone marrow stem cells demonstrated that part of the stem cells in the bone marrow differentiate to the liver lineage, so they proposed the concept "bone marrow-derived liver stem cells ".The bone marrow-derived liver stem cells which is an important source of liver stem cells extrahepatic can differentiate into hepatocyte-like cells even liver cells under certain conditions. It plays an important role in the repair and reconstruction of the liver. For now, autologous bone marrow stem cell transplantation in the treatment of cirrhosis of the liver as a new method for the treatment of cirrhosis of the liver, which is one of the world’s most cutting-edge, the most popular medical technology.The principle is that the separation and purification of the bone marrow stem cells via hepatic artery or portal vein return to the disease liver and these stem cells in the liver "settled".In clinical work, compare to the shortage of liver source and expensive of liver transplantation, autologous transplantation of bone marrow-derived liver stem cells has the easy-obtained, easy-to-vitro amplification, no tissue matching and immune rejection advantages.However, in human bone marrow mesenchymal mesenchymal stem cell is few, about every104to105single-core cell containing one MSC, and clinical application of bone marrow stem cell transplantation treatment of disease you need to have a certain quantity and high purity.Therefore, the human bone marrow-derived liver stem cells in vitro cultured,purified, amplified, identification and subpopulation of stem cells amplified analysis is particularly important.In order to induce MSC differentiation into mature hepatocytes in vitro, it is essential to have adequate stimuli for the maintenance of cellular function, such as growth hormones, cytokines, extracellular matrix or co-culture with other cell types. Currently, the unified standard of bone marrow-derived liver stem cells induced to differentiate has not yet formed at home and abroad.A variety of different cytokines used in the cultured mode, a single use of a cytokine or joint variety of cytokines. Our team research the TPO+HGF+IL-3combination culture program, the establishment a way of stable separation and purification and can effectively expand stem cell, in order to get a lot of purified bone marrow-derived liver stem cells and thus as seed cells treatment diseases related experimental basis. Hepatocyte growth factor (HGF) is the regeneration of liver-specific factor and its receptor is c-met. Especially in the case of low cell density, hepatocyte growth factor on liver stem cells migration, proliferation play a fundamental role, can effectively induce liver stem cells into liver cells, and this effect is related to HGF concentration. Interleukin-3(IL-3) and Thrombopoietin (TPO) in the bone marrow-derived liver stem cell proliferation and differentiation induction plays an important regulatory role.So far, researchers have not yet found direct identification of the characteristic surface markers of bone marrow-derived liver stem cells, bone marrow stem cell-derived lineage positioning is not very clear. But studies have shown that expression of CD90, CD105, CD117, CD184, CD326cell phenotype is the potential of liver stem cells.In this study, the target cells is isolated from human bone marrow by discontinuous density gradient centrifugation (Percoll).Bone marrow-derived liver stem cells in vitro amplification adjust the the initial amplification cell density of2.50×106/ml cultured with TPO+HGF+IL-3joint programs. Experimental group added TPO50ng/ml, HGF40μg/ml, IL-310ng/ml in high glucose DMEM medium system, the control group excluding TPO+HGF+IL-3.Observe the cell body size, shape, number of nucleoli and nuclear cytoplasmic ratio change under the microscope. Respectively,1,7and14days to calculate the total number of cultured cells and use flow cytometry to estimates expression of CD90, CD105, CD117, CD184, CD326human bone marrow-derived liver stem cell subsets, the change of the proportion and growth multiple.Create a stable separation, purification, and culture and efficient amplification of bone marrow pluripotent precursor stem cells way, and thus to obtain a sufficient amount of purified bone marrow-derived liver stem cell research as seed cells for the treatment of a variety of clinical diseases. The experimental results show that the expression of CD90, CD105, CD117, CD184, CD326human bone marrow-derived liver stem cell subsets number in the first seven days is4.94,4.89,6.70,5.72,5.03times, and14days of growth in the number of multiples is7.60,7.76,11.70,21.38,8.12times. Visible from the analysis of the data subsets in the experimental group were steady growth, which is the most rapid increase in expression of CD184subsets. This experiment confirmed that the TPO+HGF+IL-3program to stabilize the culture and amplification of bone marrow-derived liver stem cells, this combination of culture program on human bone marrow-derived liver stem cells subtypes in vitro amplification obvious effect, in which expression of CD184subsets amplification is most obvious.The characteristics and significance of this subject:TPO+HGF+IL-3programs promote the proliferation of bone marrow-derived liver stem cells, selected five subsets of bone marrow-derived liver stem cells to research the changes in number,the overall number and percentage changes, the most obvious growth subsets. In clinical application, provide an experimental basis for further human bone marrow-derived liver stem cells in the treatment of related diseases.[Materials and Methods]1.Separation, purification and culture of BMDLSC1.1Source of bone marrowBone marrow about10ml to15ml, these bone marrow is taken from between May2011to October2011, Nanfang Hospital,orthopedic surgery patients (no blood diseases).Iliac bone marrow contained seven; healthy adults anterior superior iliac spine bone marrow contained seven; eventually be included in the experiment a total of eight.1.2ReagentsPercoll separation medium (Pharmacia), high glucose DMEM (GIBCO), PBS buffer solution (newprobe), hepatocyte growth factor (HGF, Jilin), thrombopoietin (TPO, PeproTech), interleukin3(IL-3, peprotech), fluorescent antibody CD90-FITC, CD105-APC, CD117-PE, CD184-APC, CD326-PE (eBioscience), fetal calf serum (FCS, Hyclone),0.25%trypsin (Hyclone), penicillin and streptomycin.1.3Experimental Methods1.3.1the target cell separationPercoll as the separation liquid is divided into60%(1.077g/ml),50%(1.067g/ml),40%(1.056g/ml),30%(1.043g/ml) four density gradient, and fresh anticoagulation human bone marrow (10-15ml) through the the percoll liquid discontinuous density gradient centrifugation to select the density of the cellular components which is between40%-50%.1.3.2the target cell cultureStart adjusting the cell culture density of2.50×106/ml,culture environment lml, culture area of2cm2. The experimental group:high glucose DMEM medium with10%serum and TPO50ng/ml, HGF40μg/ml, IL-310ng/ml; control group cultured with10%serum-containing high glucose DMEM medium. Cells cultured in5%CO2incubator at37℃, replaced once medium every3to4day.1.3.3cell count and morphological changesRespectively,1,7and14days of culture, wipe the cell counting board and cover slips with10%alcohol. Cover glass cover on the counting board. Routine preparation of cell suspension, blowing uniform, a small sample, joined by the edge of the coverslip. Calculate the number of cells of the four cell. According to the following formula:Number of cells/ml=four grid/4×04×dilution factor.Observed the cultured cell’s body size, shape, number of nucleoli and nuclear cytoplasmic ratio changes under the microscope.2.Amplification of human bone marrow-derived liver stem cells cell subtypes2.1flow cytometry change in the number of subsetsAt the1,7and14days of culture, the adherent cells were trypsinized, counted,5×105cells in100μl were added to5μl anti-human CD90-FITC,5μl anti-human CD105-APC,20μl anti-human CD117-PE,20μl anti-human CD184-APC,20μl of anti-human CD326-PE, on ice incubated for3min,600g centrifugation for3min, the supernatant was removed, after the Army uniform cells with PBS, again600g centrifugal3min, washed twice,added1%paraformaldehyde; quickly by flow cytometry(BD FACS Caliber) detection of CD90, CD105, CD117, CD184, CD326cells percentage.2.2Data ProcessingSPSS13.0statistical software processing of data, independent sample t-test, nonparametric rank sum test; Results are mean±standard deviation; P<0.05difference was significant. [Results]1. cell morphology was observed under a microscopeEarly stage of culture, a difference under a microscope the experimental group and control group cells growth was no significant difference, the cells were fibroblast-like growth, directionality, colony vortex-shaped and flame-like, but the experimental group was more exuberant cell growth.Culture medium term, the experimental group, the original length of spindle cell gradually round colony merging into a single layer, covered the entire culture flasks, cells arranged in parallel with or swirling.Control groups cells to cultivate the late original length spindle cell remains spindle but cytoplasmic refraction, consider for aging. Late stage of culture, subculture cell growth rate than the original culture was significantly faster, longer colony formation of4-5d covered bottom.2.The total number of target cell growthFirst7days of culture, the total number of the experimental group, the total number of cells (106) for12.01±0.30, compared to5.12±0.26in the control group;14th day of culture, the experimental group, the total number of cells (106) a total of18.05±0.28in the control group compared7.05±0.23.3.Changes in the number of bone marrow-derived liver stem cells subsetsExpression of CD90, CD105, CD117, CD184, CD326BMDLSC change in the number of subsets in7days for4.94,4.89,6.70,5.72,5.03times,the first14days of growth in the number of multiples of7.60,7.76,11.70,21.38,8.12times. Various subsets of the experimental group were steady growth, the most obvious of which increase in CD184.[Conclusion]1. the steady growth of the total number of cells indicates that TPO+HGF+IL-3program is stable purification, culture and amplification of bone marrow-derived liver stem cells. This program can effectively increase the number of bone marrow-derived liver stem cells,solve the problem of insufficient number of cells encounter in the clinical application of cell transplantation,provide convenience for further liver stem cell transplantation.2. The bone marrow-derived liver stem cells can be used as an important source of seed cells in the liver tissue engineering. Bone marrow-derived liver stem cells is easy to obtain, the operation is more convenient, easy-to-survival of cultured in vitro, making it excellent seed cells in tissue engineering.3. Five bone marrow-derived liver stem cell subsets (CD90, CD105, CD117, CD184, CD326) although the growth in the culture of this program multiples, but showed a significant growth, the increase in which to express CD184subsets most obvious.CD184subsets is most sensitive to TPO+HGF+IL-3, but the exact mechanism is not clear. |
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