| Objective:To research the influence of the different concentrations of alendronate (ALN) on the ability to proliferate and to differentiate into osteoblast of human bone marrow mesenchymal stem cells (hBMSCs), and evaluate the efficiency of different alendronate solution concentrations on the differentiation rate in order to find out the most suitable concentration. Also to provide experimental evidence to the clinical application of ALN combined with BMSCs and a new thought of preventing and treating osteoporosis by traditional Chinese and western medicine.Methods:1. HBMSCs were isolated and fostered by using differential time adherent method method;2. To observe the growth,proliferate and morphologic characteristics of the original cells and their descendant by inverted phase contrast microscope day by day and darw their auxodrome.3. To get the well growing cells of fourth generation,and identify hBMSCs by detecting surface markers by morphology and flow cytometer;4. To prove the hBMSCs of this experiment have the ability of multi-differentiation by to induce hBMSCs differentiate into adipocytes,5. The well growing cells of fourth generation were divided into 3 groups: experimental group (the different concentrations of ALN, groupA-F), blankcontrol group (L-DMEM, group G) and positive control group (Dexamethasone, group H),and all of them were induced into osteoblasts.6. To detect the osteocalcin secretion by each group by Rich Internet applications (RIAs)7. qRT-PCR was used to detect the expression of LPL Runx2 and Osx mRNA of each group induced into osteoblasts or adipocytes.8. To reach the conclusion by contrasting and analyzing the statistics methods(Two sample t-tests).Result:1. The positive expression rates of CD29 and CD44 of the fourth generation cells were 100% and 99.8%, with CD31 and CD45 were 0.8% and 0.4%。It prove that this method of isolating and fostering hBMSCs could receive high-purity BMSCs,which was deteced by FCM.2. 16 days after hBMSCs were induced differentiate into adipocytes, there were many lipid droplets in the cytoplasm.AND Oil Red O Staining were positive, the expression of LPL was 83.6±4.7% detected by qRT-PCR. It proved that hBMSCs have the ability of multi-differentiation.3. At the 7 days of differentiation, a majority of hBMSCs in A-F groups continued to show a long spindle-shaped, some cells in G group began to appear morphological changes; osteocalcin expression in the culture mediums were 0.03ng/ml-0.08ng/ml in A-F groups, whereas in H group was 0.14±0.06ng/ml; 1.3%～2.5% and 1.1～2.6% cells expressed Osx and Runx2 mRNA in the A-F groups, and about 28.6±4.3% and 29.2±5.1% cells expressed Osx and Runx2 mRNA in the dexamethasone-induced group.4. At the days of 14, part of the cells changed morphology in A-F groups. In the A-D groups was 8%～12% and more cells changed morphology in E and F groups (20.7±1.4% and 23.6±1.3%), while in G group was 53.6±8.2%; compared with the first seven days, osteocalcin expression in cell culture mediums in A-F groups increased. E and F groups increased more pronounced (0.27±0.09ng/ml and 0.28±0.08ng/ml, respectively). Compared with the A-D groups, the improvement in E and F groups was statistically significant (P<0.05). In H group, osteocalcin expression was 0.46±0.13ng/ml. Compared with the first 7 days, Osx and Runx2 mRNA expression in the A-F groups were significantly increased, particularly in E and F groups (Osx mRNA:22.1±5.6% and 25.6±5.3%, Runx2 mRNA: 25.2±5.3% and 27.8±5.8%). In the dexamethasone-induced group, Osx mRNA was 55.8±10.2% and Runx2 mRNA was 53.2±9.8%.5. At the days of 21, the differentiated rate further increased in A-F groups compared with 7d and 14d. In the dexamethasone-induced group, about 84.2±10.6% cells were round. In A-F groups, the concentration of osteocalcin in cell cultured mediums was further increased. The incremental trends in cell differentiation rate and osteocalcin expression was similar. In A-F groups, Osx and Runx2 mRNA expression was further increased when induced for 21 days. More than 50% of cells positive for Osx and Runx2 mRNA in E and F groups; while in dexamethasone-induced groups, Osx and Runx2 expression were 87.5±11.5% and 85.9±10.2% respectively.6. In the negative control group, there was no change in cell morphology, osteocalcin expression and Osx and Runx2 mRNA expression.Conclution:1. The stable and uniformal hBMSCs could be obtained by differential time adherent method;2. The ALN could promote the proliferation of the hBMSCs and the differentiation of hBMSCs into osteoblast, and 1×10-6 mol/L is the most suitable concentration. |
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