| Previous studies showed that bone marrow mesenchymal stem cells (BMSCs) had many biological characteristics, such as easy to separation and amplification, lower immunogenicity, and also showed the ability of differentiation as "seed" cells. BMSCs have the functions of variety of factors secretion by autocrine or paracrine adjustment, and that of inflammatory reaction. Many experts have been focus on the effects of BMSCs, such as to repair injured ischemic myocardial cells, osteocyte and to promote blood vessel regeneration, etc. A number of studies have reported that the mechanisms of atherosclerotic plaque formation were related with inflammation-fibroplasia of artery which response to damaged vascular endothelial cell and endomembrane. So it will be the evidence to treat atherosclerosis with BMSCs because of the characteristics of BMSCs. Eliminating Sputum and Removing Stasis Formulation, the traditional Chinese medicine, Semen Trichosanthis, Red Yeast Rice, RongstamenOnion Bulb and Panaxnotoginseng are its effective component. Acute toxicity test have been finished and the animal model of atherosclerosis in rat have been made by our research group. The results indicated that Eliminating Sputum and Removing Stasis Formulation could significantly decrease cholesterol level, stabilize the plaque, and inhibit the progress of atherosclerosis without any side effect. This study aims to investigate the effects of Eliminating Sputum and Removing Stasis Formulation on BMSCs and afford the experimental fundation for the application in future studies.ObjectiveTo observe the effects of Eliminating Sputum and Removing Stasis Formulation on proliferation, apoptosis, migration and vascular endothelial growth factor (VEGF) secretion of rat bone marrow mesenchymal stem cells (BMSCs) in vitro, and explore the potential mechanisms of BMSCs migration.MethodsBMSCs were extracted form the hind limb of rat with bone marrow adherence method. BMSCs were cultured. P3-P6 cells were used for cellular morphological observation and identification by inverted microscope. The orient induction of BMSCs into adipogenic and osteogenic, and verified those cells after differentiation with oil red O and Alizarin red. Identified the BMSCs surface specific antigens expression of CD29, CD34, CD45 and CD90 by flow cytometry and than calculated positive expression rate. Different Incubation time (12h、24h、36h、48h、60h、72h) and different concentration of Eliminating Sputum and Removing Stasis Formulation (10μg/L、25μg/L、50μg/L、100μg/L、 150μg/L) were conducted on BMSCs to detect the proliferation effects (Celll Counting Kit-8 (CCK-8)cell proliferation testing kit) and than selected the optimal concentration of Banxiao and cultured time. The migration rate of BMSCs was measured by Transwell in different concentrations and optimal time and also apoptosis of BMSCs was detected by flow cytometry. The cells were cultured with optimal concentration and time and detected the expression of CXCR4 protein by Western Blot. The supernatant of BMSCs (different concentrations and optimal incubation time treated by Eliminating Sputum and Removing Stasis Formulation) were collected respectively. The ELISA assay was subjected to detect the VEGF expression.Results1. Isolation, culture of identification to cultured BMSCs in vitroBMSCs can be passaged after 14 to 21 days cultured by the whole bone marrow adherent method, during which P3-P6 generations are of higher purification and activity. BMSCs are in the shape of spindle or polygonal, spiral shaped arrangement with more cells. Cells can be induced to differentiation after cultured by special medium. Identified by flow cytometry, the expression of CD34, CD45 on BMSCs was negative (2.47%, 1.94%), while CD29, CD90 was positive (98.23%,98.69%) that are the characteristics of BMSCs.2. The effects of Eliminating Sputum and Removing Stasis Formulation on proliferation and apoptosis of BMSCsThe growth curves were draw based on OD values measured at different time points when different concentrations of Eliminating Sputum and Removing Stasis Formulation contents co-cultured with BMSCs. Compared with the control group of curves, the curve of drug concentration lOug/L,25ug/L and 50ug/L had showed the proliferation effect. The curves of 100ug/L and 150ug/L group had showed inhibit proliferation effect which under the control group curve. In the 50ug/L group when the incubation time was 24h, 36h,48h,60h,72h,25ug/L incubation time at 48h,60h,72h, lOug/L incubation time at 60h, and 72h, cell survival rates were higher at the same time point compared with the control group, the difference was statistically significant, P<0.05; and the cell survival rate of 50ug/L group was significantly higher than at the same time point of 10ug/L and 25ug/L concentration group when BMSCs cultured after 36h, P<0.05. The rusults showed that the proliferation of BMSCs get fastest after cultured for 48h with the concentration of 50μg/L. Flow cytometry showed that anti-apoptotic effects were resulted from 25μg/L,50μg/L Eliminating Sputum and Removing Stasis Formulation cultured for 48 h, there was a significant difference compared with the control group (P<0.05).3. The effect of Eliminating Sputum and Removing Stasis Formulation on migration of BMSCsTranswell showed that the number of BMSCs migration was increased significantly in the group of 10μg/L,25μg/L,50μg/L and 100μg/L at incubation 48h (p<0.05). It was the most significant at 50μg/L after incubation 48h. Under such conditions (50μg/L,48h), the expression of CXCR4 protein of BMSCs is higher than the control group with statistical significance (P<0.05) by Western Blot.4. The effect of Eliminating Sputum and Removing Stasis Formulation on VEGF secretion of BMSCsELISA indicated that the highest secretion of VEGF was obtained from the cell treated on concentration of 25ug/L,50μg/L incubated 48h [(406.22±10.71) pg/ml, (410.44±12.15) pg/ml] compared with the control group (387.56±10.25) pg/ml were higher than the control group with statistical significance, P<0.05.ConclusionsEliminating Sputum and Removing Stasis Formulation (10μg/L,25μg/L, 50μg/L) can bring the effects on proliferative promotion, apoptotic inhibition on BMSCs and facilitate the secretion of vascular endothelial growth factor in vitro when drug concentration was 25μg/L,50μg/L. Besides, Eliminating Sputum and Removing Stasis Formulation can promote BMSCs migration which related with CXCR4 protein expression. |
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