| Objective:To observe the effect of centrifugal force stimulates on different stage of mouse bone marrow mesenchymal stem cells(BMSCs) to chondrocytes in micromass culture conditions. To explore the mechanism of the centrifugal force and the Notch signal pathway in the process of cartilage differentiation. Methods:The BMSCs were isolated from the KM-mouse femur and tibia bone marrow. After subculture and proliferation,flow cytometry identify the P3 cell. Then make the cell micromass as the number of 1×106 per one. Accroding to the different treatments, we divided our research into six groups:Group A: the centrifugal force stimulates for the first week.Group B: the centrifugal force stimulates for the second week.Group C: the centrifugal force stimulates for the third week.Group D: the centrifugal force stimulates for the last week.Group E: the control group, the cell micromass at common culture condition.All of the cells were cultured with induction medium.Use the Eppendorf 5920 R constant temperature centrifuge to provide 37℃, 200 G, 2h/d relative centrifugal force.After centrifugal force stimulate for each group,the cell proliferation efficiency of each group was tested by CCK-8 and PCNA.RT-PCR was used to test the expression of Sox9,Col-Ⅱ,Notch1,Hey1 and Hes1 after centrifugal force stimulate for each group and the control group.After 28 days,test the expression of Sox9,Col-Ⅱ,Notch1 and Hey1 of each group.The Col-Ⅱ protein in the culture medium of each group at different time points was tested by Elisa.Safranine O staining was used to test the ECM and immunofluorescence technique was used to test the Col-Ⅱ of each group after 28 days. Results:1.In the beginning of incubation,the cells were quasi-circular,after 24 hours the cells attached to the wall, After subculture,the cells had quick proliferation, and tuned to long shuttle-shape, arranged in or whorls. Testing results of flow cytometry: CD45 was strongly positive expression,CD11 b,CD34 and CD29 expression were strongly negative expression.The cells were bone marrow mesenchymal stem cells and highly purified.2. Cell proliferation rate detection: Cells proliferation of group A and B was obvious compared to the control group.Cells proliferation of group C and D was not differ significantly compared to the control group.3. RT-PCR test:After 28 days, compared to the control group,the expression of Sox9 and Col-Ⅱ had the similar trend, the levels of Sox9, Col-Ⅱ of group B and C increased significantly,the group A decreased significantly, while there was no significant different between group D and control.The expression of Notch1 and Hey1 had the similar trend, the levels of group B and C decreased significantly and group A and D increased significantly.There was no significant different between each group of Hes1.At 7 day, compared to the control group, the levels of Sox9, Col-Ⅱ of group A was lower,while the levels of Notch1 and Hey1 was higher.At 14 day, the levels of Sox9, Col-Ⅱ of group B was higher,while the levels of Notch1 and Hey1 was lower compared to the control group.At 21 day,the expression of Sox9,Col-Ⅱ,Notch1 and Hey1 in group C had the similar trend to group B. There was no significant different between group B and group C of each gene all of above.4. The concentration of Col-II in the culture medium showed an increasing trend with the culture time. Compared to the control group,the Col-II in group A had the slowest growth and the lowest concentration after 28 days.Group B had the fastest growing and the highest concentration after 28 day.Group C had a bit slower growth and the similar concentration compared to group B. There was no significant different growth and final concentration between group D and control.5.The ECM and Col-Ⅱ of each group:group B had the most expression,while groupA had the most cell. Conclusion:Different stages of centrifugal force have the different effects on the differentiation of mesenchymal stem cells into cartilage. The second week of cartilage differentiation is the best time of centrifugal force intervention while the most cartilage matrix and proliferation.Early centrifugal force intervention mostly can activate Notch signaling pathway, which inhibits the differentiation of cells into cartilage.Delayed centrifugal force may promote the formation of cartilage by inhibiting the Notch signal. |
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