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

Human mesenchymal stem cells (hMSCs) are self-renew cells present in adult bone marrowpostnatal stem cells, hMSCs has transgermal potential, and have the capacity to differentiate into a variety of tissue types of mesodermal lineage including bone, cartilage, cardiomyocyte, neurons.Evidence suggests that these cells might also be able to differentiate along the ectodermal and endodermal lineages, but whether full differentiation down these pathways is actually achieved is still under investigation. While their ability to differentiate is important,other significant functions of these cells include regulating hematopoiesis, secreting factors that aid wound healing by preventing apoptosis and stimulating endogenous cellular repair, and controlling inflammatory and immunological reactions. The availability of bone marrow and the ability to isolate and expand hMSCs in vitro make these cells an attractive candidate for medical research and clinical application. However,many issues will have to be further investigated to more fully understand the behavior of the stem cells in these applications. In particular,in vitro biological characteristics of cells is the foundation of basic and clinic research of MSCs.Ecdysterone(B-ecdysone) is a kind of hormone of hexapod to modulate metabolic regulation. It was researched by entomologist in early time. Hexapod scrustacean and arthropod have callous corneous layer. They secrete ecdysterone to exuviate the corneous layer regularity and grow new layer. This process is called ecdysis. But it was found that ecdysterone exists in vegetable kingdom more than in animal kingdom. The researchful application of ecdysterone is mostly depend by distill of plants by now. Ecdysterone is indispensable to vertebrate.It's main mechanism is to:1)promote protein synthesis,2)modulate glycometabolism,3) modulate lipid metabolism,4)modulate gene expression,5)improve immune function,6)act to central nervous system,7)act to cardiovascular system,8)promote concrescent.Because Ecdysterone has various activities, including stimulating protein synthesis, promoting carbohydrate and lipid metabolism, alleviating hyperglycemia and hyperlipemia, immunologic modulation, and protecting endothelial cells from apoptosis and inducing their proliferation.Ecdysterone can obviously promote wound healing in rabbitsand .EDS has a proliferative effect on the growth of epidermal stem cell in vitro. It is a major component of several Chinese herbal medicines, such as Achyranthes bidentata BL. and Cyanotis arachnoidea C. B. CLARKE.various activities of Ecdysterone in human being,especially stimulating protein and RNA synthesis. So we considered that it may enhances proliferation and differentiation of hMSCs.To isolate, culture and identify the human bone marrow mesenchymal stem cells. hMSC were isolated from human bone marrow by gradient centrifugation. The expression of integrins CD44, CD105,CD34 and CD29 were examined by immunocytochemical method. Cell surface markers CD44,CD34 and CD105 were tested by flow cytometer.The 3rd,5th and 10th generation well-grew cells were digested into cell suspension and inoculated. Cells growth curve was drawn taking amount of cells as Y-axis and time as X-axis.1.Morphological observation of hMSC:Under inverted microscope, the hMSC began to adhere to the wall within 12 hours. Cells were spindle-shaped and presented active proliferation in primary and passage cultures. hMSC began to proliferate and become into Fusiform shape, polygon and irregularity at day 3.hMSC concentrated in the center of colony and covered the bottom of culture flask at day 14.2.The cell surface markers CD44,CD29,CD105 were positive and CD34 were negative.3.The flow cytometry shows that1.64% of the cells are CD34 positive,92.61% of the cells are CD44 positive and 91.77% of the cells are CD44 positive.4. Growth curve of hMSC:hMSC were in latency in 3 days,converted into growing period after 3 days and entered into s tationary phases after 7 days.1.hMSCs can separate in vitro, purify culture, grow stableand serial passage.2.The immunocytochemical method and flow cytometry shows that the cultured cells are neither hemopoietic stem cells nor fibroblasts but the hMSCs that have not differentiated.3.After serially subcultivated hMSC appear to be aging. This study was to investigate the effects of Ecdysterone (EDS) on the proliferation of human bone marrow mesenchymal stem cells (hMSC) in vitro.The various concentrations of EDS (10μg/mL,25μg/mL,50μg/mL and 100μg/mL) were added in the culture system for hMSC and the common culture medium for hMSC was used as control. Then the cell number viability were analysed by MTT assay and the cell cycle was examined by flow cytometry analysis. The various concentrations EDS group(10μg/mL,25μg/mL,50μg/mL and 100μg/mL) and control group of 5th generation cells were digested into cell suspension and inoculated. Cells growth curve was drawn taking amount of cells as Y-axis and time as X-axis.1.The results of optical density (OD) showed that there were significant differences between the EDS adding groups and the control group (P<0.01);and the OD of 25μg/mL group showed the more obvious efficiency(P<0.01).However, there were no significant differences among the 10μg/mL,50μg/mL and 100μg/mL group (P> 0.05).2.Growth curve of hMSCs:EDS 25μg/mL group has stronger proliferative activity than the other groups, and the EDS adding groups has stronger proliferative activity than the control group.3.The results of flow cytometry analysis showed that the ratio of S-period and G2M-period cells were obviously more than other groups, and proliferation index (PI)of marrow mesenchymal stem cells in EDS25μg/ml group was higher than that of the control group.The centain concentration range of EDS has a proliferative effect on the growth of hMSC in vitro and the most obvious efficient concentration is 25μg/mL. But the proliferative effect could not be enhanced by the increasement of concentration.To study the effects of Ecdysterone on differentiation of hMSC.25μg/mL of EDS were added in the culture system for hMSC and the common culture medium for hMSC was used as control. The surface maker of MSC CK19 and CK10 were examined by immunocytochemical method, CK19 were tested by flow cytometer. The result of flow cytometer and immunocytochemical method show that whether MSCs were induced in vit ro respectively to epidermic like cells by EDS.Calcified nodules were observed using alizarin red S staining. The result of alizarin red S staining differentiation of hMSCs into osteoblasts cells in vitro by using EDS.1.The cell surface markers of CK19 and CK10 in both 25μg/mL EDS group and control group were negative.2.The flow cytometry shows that1.64% of the cells are CD34 positive,92.61% of the cells are CD44 positive and 91.77% of the cells are CD44 positive.3.Calcified nodules were found in25μg/mL EDS group,but didn't found in the control group.25μg/mL EDS can not influence the differentiation of epidermic cells from hMSC, but 25μg/mL EDS enhances osteogenic differentiation of hMSC.