Effects Of Ecdysterone On Biological Characteristics Of Human Umbilical Cord Mesenchymal Stem Cells In Vitro

BackgroundSince mesenchymal stem cells by Friedenstein found in 1966 that starting from the bone marrow,more and more studies show that mesenchymal stem cells under certain conditions have the ability to differentiate into the inner, middle and outer of three germ layers tissue types including bone cells,chondrocytes, fat cells, endothelial cells, liver cells, myocardial cells and other potential of cells. Mesenchymal stem cells can repair a wide range of participating organizations, as tissue engineering and stem cell research focus.Bone marrow-derived mesenchymal stem cells in which are easy to culture,genetic background is relatively stable and not affected much to the differentiation, can be used for clinical cell and tissue replacement therapy.But with the deepening of the study researchers found that the proliferation and differentiation potential of bone marrow mesenchymal stem cells from the adult animals falling increases with age. We get the mesenchymal stem cells from donor using puncture collection, because the disease reasons, patients often infected, weak constitutions and other factors also limit the autologous bone marrow mesenchymal stem cell transplantation widely used. So to find a new source of mesenchymal stem cells stem using for cell research is a hot spot at home and abroad. In 1991,McElreavey first reported that WJ from human umbilical cord tissue to be a kind of isolated and cultured fibroblast-like cells with high differentiation potential which can be divided to multiple directions.Subsequently, several researchers were also reported isolated MSCs from the WJ.By a large number of experiments in vitro confirmed that under certain conditions it also has multipotent differentiation capacity including osteogenic cartilage cells,fat cells, muscle,vascular endothelium cells,nerve cells and many other cells.Compared with other sources of MSCs,especially the gold standard of BM-MSCs,umbilical cord stem cells have great advantages,such as the first, the wide variety of sources,drawing convenience for those without pain;the second,the relatively pure,pollution,fewer opportunities;the third,the content is rich and also no age effect,the more primitive,proliferation and differentiation ability,and biological properties of stable,adequate for the experimental and clinical cell source.Therefore,the umbilical cord mesenchymal stem cells are expected to become the ideal alternative source of stem cells, as recent basic research and clinical research focus.Both researchers and the public hopes by mesenchymal stem cell research to cure much disease such as refractory wound healing, multiple organ damage, myocardial infarction and other traditional methods of treatment are ineffective.In recent years,there are more and more countries are joining the study,although mesenchymal stem cell therapy in clinical effect has not yet been fully confirmed.However,there are many challenges and critical issues to be resolved before the treatment of mesenchymal stem cells in order to truly from the laboratory and clinical application.In particular,biological characteristics research of mesenchymal stem cells is the essential and the basis for clinical application. Ecdysterone (EDS),is a process of regulating insect molting hormone,widely found in many plant groups and animal taxa,a structure similar to estradiol.The experiments confirmed that it can effectively promote a variety of mammalian tissues and organs Nucleic acid and protein synthesis, and glucose and lipid metabolism. Because of such extensive source material, biological characteristics of complex, has attracted many scholars to join this research.Otaka reported that ecdysterone in vivo or in vitro can both quickly stimulate protein synthesis in rat liver.In vitro, ecdysterone able to promote the synthesis, thus enhancing microsomal and ribosomal protein synthesis.Yoshida T reported that ecdysterone had the metabolism on glucose in the regulation effect,pre-peritoneal injection of ecdysterone is able to resist intraperitoneal injection of glucagon and intravenous injection of insulin resistance caused by hyperglycemia.Syrov VN reported that ecdysteroid liver protective effect of alcohol. With the deepening of the study, the biological ecdysterone more features will be revealed.EDS previous findings by our group can promote the proliferation of human umbilical vein endothelial cells, promote the healing of skin wounds, the promotion of skin and bone marrow mesenchymal stem cell proliferation and so on.New research shows that, under certain conditions steroid dexamethasone has the aility of inducing hUCMSCs to differentiate into bone cells and expression of osteopontin, sialoprotein, osteonectin and bone markers and also can effectively induce mesenchymal stem cells into osteoblasts.Ecdysterone demonstrated biological effects,suggesting that its umbilical cord to promote mesenchymal stem cell proliferation and induction of differentiation and so to a certain extent, for this research has broad application prospects.Part I Biological characteristics of human umbilical cord mesenchymal stem cells in vitroPart I Isolation,Cultivation and identification of human umbilical cord mesenchymal stem cellsObjectiveTo isolate,culture and identify the human umbilical cord mesenchymal stem cells (human umbilical cord mesenchymal stem cells, hUCMSCs)and provide seed cells for the future experiments.MethodsThe hUCMSCs were isolated from human umbilical cord by digestion with collagenase and tissue culture method.After serial subcultivation in vitro,the stem cells were passaged.Morphologic appearance of hUCMSCs was observed under an optical under an optical microscope and atomic force microscope.Cell surface antigens CD29,CD34,CD44,CD45,CD90,D105were measured by flow cytometry. The osteogenic and adipogenic differentiation was tested and evaluated by specific staining methods.The proliferation rate was measured by MTT assay.Results1. Morphological observation of hUCMSCs:Inverted microscope, stem cells from enzymatic digestion, primary inoculated cells after 24 h shows a small amount of adherent, spindle-shaped appearance and the short rod and visible nuclei.Most of the cells cultured about 5d were protruding spindle, flat-shaped growth, more significantly in the nucleolus.Cultured about for10d,the cells become typical fibroblast morphology, when cells reached 80% confluence to digestion and passage.Stem cells witch ubcultured to P3 apparent a single colony.From the primary culture of view, collagenase digestion than tissue culture method is more efficient, which are about lOd can be passaged then the cells from tissue culture can be passaged 14d.There was no significant between Sexual difference in morphological changes and the passage.2.Flow cytometry detection:CD34 positive rate was 7.54%,CD45-positive rate was 5.35%,CD29-positiverate was 98.45%,CD44-positive rate was 97.83%, CD90-positive rate was 97.36%,CD105-positive rate was 99.20%.3. hUCMSCs induced differentiation in vitro 3.1 hUCMSCs bone induction and functional identification of the morphological changes:hUCMSCs are inducing Osteogenesis for 5d about spindle cell morphology changed gradually from a triangle or polygon, etc.Observed under high magnification microscope, the cytoplasm contains granular material visible.Induction Osteogenesis of 10d, many small black particles are visible in the cytoplasm and outside of the cytoplasm,and also the color lighter nuclei, cytoplasm pale deeper.Some stem cells showed stacked like growth and the central shows a similar nodular structure after 3W intensive cell growth. Alkaline phosphatase staining showed strong positive, in line with the characteristics of osteoblasts.ALP and Alizarin red staining showed strong positive, in line with the characteristics of osteoblasts.hUCMSCs in vitro osteogenic has the differentiation potential.3.2 hUCMSCs adipogenic and functional identification of the morphological changes:the cells with adipogenic for about 7day changed gradually from short spindle to spindle cell morphology;About 14d the cells in the induction changed into oval, round;20d cytoplasm can be seen when the lipid droplets by oil red "O" staining was strongly positive.hUCMSCs in vitro could differentiate into the fat cells.4.hUCMSCs growth curve:hUCMSCs S-shaped growth curve,hUCMSCs after inoculation of 1 to 2 days for potential adaptation period,from day 3 onwards cells begin to proliferate and enter the logarithmic growth phase,reached a peak 5 days later into the plateau.HUCMSCs based on the growth curve showed that the population doubling time is about 24h.Proliferation curves indicate that the experiment indicating that the P3,P7cells had no significant difference in proliferation rate.Conclusion1. hUCMSCs getting from enzymatic digestion and tissue culture in vitro isolated and purified culture, cell growth and a stable, continuous passage. Tissue culture method for primary culture time was 14～16d,a longer incubation time and by collagenase digestion method can quickly obtain the growth of a large number of adherent MSCs.But the operation is simple, can better maintain the cell viability, and also greatly reduce the time of primary culture.2. By flow cytometry testing,hUCMSCs uniformly high express the stromal cell markers,such as,CD29, CD44, CD90, CD105,did not express hematopoietic signs CD34, CD45.Our results show that hUCMSCs are the same as other organizations phenotypes with flow cytometry.3. Cells were treated with osteogenic and adipogenic induction medium induced by ALP staining and alizarin red staining confirmed that differentiate into bone cells, confirmed by oil red O staining to differentiate into fat cells.Our experiments confirmed that hUCMSCs have the capacity of multipotent differentiation.4. There was no significant difference between 3, and 7 third-generation cell proliferationPartⅡBiological characteristics of human umbilical cord mesenchymal stem cells following cryopreservationObjectiveObservation of cryopreserved umbilical cord mesenchymal descendants of stem cells in human umbilical cord mesenchymal stem cells (hUCMSCs) recovery rate and the value-added activities, to research the feasibility of long-term storage with liquid nitrogen.MethodsWith reference to the previous method to obtain human umbilical cord mesenchymal stem cells cryopreserved using the modified method of stages,5 months after the recovery of cell culture, observed cell survival rate of all treatments and the ability of cell proliferation MTT assay differences, streaming Cells detected by frozen cell phenotype changes.Results1.Morphological characteristics of the recovery hUCMSCs resuscitation rate of frozen cells was (86.40±1.34)%.2h after resuscitation began adherent cells are gradually transformed into spindle and polygonal. After about a week to recover, the third generation cell morphology had no significant difference.2.Flow cytometry recovery hUCMSCs mesenchymal stem cell line with phenotypic characteristics. MTT test cryopreservation does not reduce the mesenchymal stem cell proliferation.ConclusionFrozen cells by detecting the survival rate of experimental groups by flow cytometry, cell surface markers and cell cycle characteristics did not change, indicating that the liquid nitrogen freezing can be stored for a long mesenchymal stem cell; In addition, the proper choice of logarithmic growth Cryopreservation of cells, and frozen cell concentration should reach 1±106/ml also plays a key role.PartⅡEffects of ecdysterone on proliferation of human umbilical cord mesenchymal stem cells in vitroObjectiveThis study was to reseach the effects and mechanism of EDS the on proliferation of hUCMSCs in vitro.MethodsThe third generation of hUCMSCs were cultured in basic medium, respectively(low glucose DMEM,10% fetal bovine serum, 100U/m Penicillin/ streptomycin,4mmol/L L-glutamine) with different concentrations(0,25,50,100,150, 200μg/ml) EDS to intervene, were the first line of 1,3,5,7-day MTT cell proliferation assay.After that EDS to set the same group intervention training hUCMSCs 7 days,cell growth curve,observe and compare the differences in cell proliferation.Cells were measured cell cycle in different groups,the proportion of cell proliferation observed.Line statistical results.Results1. MTT assay showed that EDS intervention by the proliferation of cultured hUCMSCs than that of control group were significantly different (P<0.05),in which EDS 100μg/ml,150μg/ml and 200μg/ml cell activity in the three groups showed no significant difference (P>0.05),compared with other experimental groups was statistically significant (P<0.05).2. The cell growth curve of the experimental group also showed that EDS can effectively promote mesenchymal stem cell proliferation.EDS of 100μg/ml proliferation in experimental group the most obvious effect,but EDS 100μg/ml, 150μg/ml and 200μg/ml three groups were not significantly different.3. Flow cytometry showed the cell cycle:EDS 100μg/ml,150μg/ml and 200μg/ml three groups of cells in S phase cells the largest share,compared with other experimental groups had significant differences,while three groups of cells The proliferation index was also significantly higher.ConclusionIn vitro conditions,this experiment confirmed that EDS has a certain concentration range for hUCMSCs proliferation and the role of a certain concentration dependent,when the EDS promoting proliferation of the concentration of 100μg/ml best results,we suggest that EDS is mainly through Change the cell cycle to promote cell proliferation. Part III Effects of ecdysterone on differention of human umbilical cord mesenchymal stem cells in vitroObjectiveTo study the effects of ecdysterone on osteogenic differentiation of hUCMSCs.MethodsHuman umbilical cord mesenchymal stem cells were isolated, cultured and identified,then the 5th generation of mesenchymal stem cells were induced in the basic medium with different concentrations of dexamethasone and EDS to intervene, respectively, on day 7 MTT cell proliferation assay; In addition, the same group set the culture for 4 weeks after induction, calcium by alizarin red and cobalt alkaline phosphatase staining ability of osteogenic differentiation identified.Calculate the percentage of positive cells.Results1,MTT assay showed a range of EDS with the promotion of cell proliferation, and dexamethasone can inhibit cell proliferation.2,Alizarin red and alkaline phosphatase staining of calcium cobalt method showed a higher concentration of both EDS and dexamethasone can induce hUCMSCs calcified nodules, bone staining.ConclusionAt a concentration of 200μg/ml,EDS have the bility of inducing osteogenic differentiation of hUCMSCs,but significant inhibition of cell proliferation.EDS through the preliminary view that the role of estrogen receptors. EDS200μg/ml group composed of bone formation than EDS100μg/ml significant difference,indicating that the induction of bone formation with a certain concentration dependent manner...