Effect Of Tanshinone â…¡A Sulfonate On The Differentiation Of Rabbit BMSCs Into Chondrocytes And The Effect Of Cell Density On Induction

BACKGROUND:Bone marrow mesenchymal stem cells (BMSCs) is a bone marrow hematopoietic cells in the structural and functional support cell.Representing the ratio of bone marrow cells in the bone marrow is trace.It increases with age and the number of cells is significantly reduced with typical fusiform, whirlpool growth, with strong differentiation potential.Chondrocytes (Chondrocyte) is the only cell type in cartilage tissue.Chondrocytes generate and maintain cartilage matrix (including collagen and proteoglycans).Limited proliferative and survival density requirements of chondrocytes is the main reason to limit for the repair of articular cartilage defects and in vitro expansion, and Human articular cartilage injury repair is still a major problem today.Objective:Separate cells to subculture and observe its biological characteristics utilize multiple detection methods.Verify the amplification effects of Tanshinone IIA sodium to BMSCs and chondrocytes in vitro and find out the optimal drug concentrations in both cell growth promoting for further experiments.Methods:Method of bone marrow adherent separation and Two-step enzymatic digestion were used to extract BMSCs and chondrocytes.Observe the cell biology shape under an inverted phase contrast microscope and identify with correlation detection.Preparatel5 different concentrations of sodium tanshinone IIA solution with geometric dilution.Separately cultured newborned rabbits’BMSCs and chondrocytes and two types of cells in 1:1 mixture of the two cells then explore the best drug concentration.Results:Newborned rabbits’BMSCs and cartilage cells were fusiform morphology and short spindle.Correlation tests were all identified as target cells.Tanshinone IIA sulfonate was used for incubating 1:1 ratio of mixed cells had showed a growth.The optimum concentration is 50ug/ml and in the interval 37.5ug/ml cells to 62.5ug/ml when cultured alone.So 50ug/ml was the best culture concentration for further experimental studies.Conclusion:Newborned rabbits’BMSCs and chondrocytes extracted with whole bone marrow adherence method and two-step enzymatic digestion were feasible.Tanshinone IIA sulfonate optimum concentration should be 50ug/ml.Objective:Find out the role of Tanshinone IIA sulfonate on newborned rabbits’BMSCs induced transformation into chondrocytes.Methods:Establish the group with replacing medium only for the negative control grouP and the best tanshinone IIA sodium concentration for induced experimental group and TGF-β1-induced cartilage systemfor the positive control group.12-well plates were for culture plate, set climb piece" tobottom of the hole then induced for 7 days,14 days,21 days.Then remove the pieces climbing and conduct Safranin O staining, toluidine blue staining, type Ⅱ collagen immunohistochemistry, FDA/PI fluorescence detected by RT-PCR and quantitative detection of proteoglycans and expression of type Ⅱ collagen. Verify the function of Tanshinone IIA sulfonate inducing transformation newborned rabits’BMSCs to chondrocytes.Result:newborned rabits’BMSCs in negative control group did not change trait significantly.No positive performance of various detection; experimental group and TGF-β1-induced system cartilage morphological changes were found.Gradually fusiform changed into short spindle or polygonal of cells.A variety of detection performance were positive, and the longer in the induction process,the more cartilage cells were producted.However, TGF-β1-induced cartilage system was significantly more than the experimental group.RT-PCR analysis showed that the experimental group and the TGF-β1-induced system group had the expression of cartilage proteoglycan and collagen expression of type Ⅱ. Experimental group was less than TGF-β1-induced cartilage system group.Conclusion:Tanshinone IIA sodium has the effect of promoting newborned rabits’BMSCs induced transformed into cartilage cells.Objective:To study the role of cell density induced transformation BMSCs into chondrocytes.Methods:Prepared 12-well plates for different densities of newborned rabits’ BMSCs and divided into three groups, low-density group, medium-density and high-density group group.Use the TGF-β1-induced cartilage induction system for transformation experiments, by periodic replacement of the culture medium. Cultured for 14 days after the induction then take photos of cell morphology, type Ⅱ collagen immunohistochemistry, FDA/ PI fluorescence development and RT-PCR and other testing.Verify the affect of cell density induced transformation process.Results:The three groups significantly induce BMSCs morphology after 14 days,and Type Ⅱ collagen immunohistochemical staining were positive.Aggrecan and type Ⅱ collagen RT-PCR showing most expression of maximum density group.Conclusion:Cell density is a factor in induced transformed BMSC into chondrocytes.