Experimental Study Of The Effects Of CD105-ABBS On Capturing Synovium Derived Mesenchymal Stem Cells

Part Ⅰ:Isolation of CD105+SMSCs and evaluation of chondrogenic capacity of CD105+SMSCsObjective:To compare chondrogenic capacity of CD105+SMSCs and CD105"SMSCsMethods:SMSCs were divided into two groups:CD105-SMSCs and CD105+SMSCs. CD105SMSCs were enriched by Fluorescence-activated cell sorting (FACS). The proliferation of cells in both groups was detected by WST-1assay at day3,7and14after seeding. Immunohistochemistry was prepared to investigate the expression of type Ⅱ collagen of both groups at day14. The expression of type Ⅱ collagen, aggrecan, and sox9mRNA of both groups was tested by RT-qPCR in both groups at day14.Results:Morphology of cells in both groups showed spindle-shape or stellate-shape. Results of immunofluorescence showed more positive expression in CD105+SMSCs group. The results(od value) of WST-1assay in CD105SMSCs and CD105+SMSCs were0.329±0.0120VS0.376±0.012at day3,0.524±0.017VS0.581±0.019at day7,0.658±0.032VS0.702±0.099at day14, P<0.05at each day with a statistical significance. Expression of type II collagen of cellular matrix was stronger in CD105+SMSCs group than in CD105"SMSCs. The results of RT-qPCR(2-△△CT) in CD105-SMSCs and CD105+SMSCs were1VS2.937±0.899(col Ⅱ),1VS10.676±2.276(sox9)、1VS4.282±0.463(aggrecan), P<0.05with a statistical significance.Conclusion:The process of enriching CD105+SMSCs by using FACS showed no obvious adverse effects on chondrogenic capacity of CD105+SMSCs. Compared with CD105"SMSCs, CD105+SMSCs were stronger in chondrogenic capacity. Part Ⅱ:Experimental research of the value of CD105-ABBS on capturing SMSCsObjective:Evaluation of the value of CD105monoclonal antibody-biotin-avidin binding system on capturing CD105+SMSCs and its influence on chondrogenic capacity of SMSCsMethods:ABBS and CD105-ABBS were prepared on cell slides respectively. Slides were transferred into a same culture dish which contained SMSCs suspension. and then the culture dish were put into a cell incubator. Thirty minutes later, slides were taken out of the culture dish, and vibrated for a while to wash away unattached cells. The differences of the number of attached cells were comparied, so as proportion of CD105+SMSCs. Immunohistochemistry was prepared to investigate the expression of type Ⅱ collagen in both groups. The expression of type Ⅱ collagen, aggrecan, sox9mRNA of both groups was tested by RT-qPCR at day14.Results:The number of attached cells in ABBS and CD105-ABBS was20.33±5.43VS40.67±7.54, P<0.05with a statistical significance. Proportion of CD105+SMSCs of attached cells in ABBS and CD105-ABBS groups was (47.67±7)%VS (72.33±11.93)%, P<0.05with a statistical significance. Expression of type Ⅱ collagen of cellular matrix was stronger in CD105-ABBS group than in ABBS group. The results of RT-qPCR(2-△△CT) in ABBS and CD105-ABBS were1VS1.835±0.369(col Ⅱ).1VS2.808⒈0.189(sox9)、1VS3.146⒈0.552(aggrecan), P<0.05with a statistical significance.Conclusion:CD105-monoclonal antibody-biotin-avidin binding system can be used to capture CD105+SMSCs, which provides efficient method for situ regeneration in vivo and tissue engineering construction in vitro.