Comparative Analysis On The Effect Of Shaoyangshenggu Formula On Proliferation Of Dedifferentiation Chondrocytes And Its Collagen Type â…¡ Expression

object:This study is to analysis the effect on “shaoyangshengguformula” ruled by the theory of “shaoyang dominant bone”and”reconciliationShaoyang” on dedifferentiation chondrocyte.Using a comparative analysis atthe cellular level to explore the effect of “shaoyangshenggu formula” ondedifferentiation chondrocyte by microscope\CCK-8\immune.To provideexperimental basis and theoretical reference of research on TCM affectdedifferentiation chondrrocyte, to provide feasibility and possiblemechanisms for treatment of cartilage damage, to provide a reference for thenext step to continue to study and learn" shaoyang dominant bone" theory.Ways:1. The isolating\culturing\identification system established by the way ofmechanical separation and enzymatic digestion.Using the Collagenase Ⅱ and Trypsin to digest femoral condyle cartilagefrom one week-old rats times to obtain the cell suspension,the cellsuspension was collected and cultured in DMEM culture medium containingfetal.Observe and record the morphology,size and growth of chondrocytedaily by inverted microscope. The chondrocyte was identified by toluidineblue staining and collagen type Ⅱ staining.2.Study on proliferation\cell cycle and apoptosis of chondrocyte by CCK-8. Put the isolated chondrocytes into DMEM medium containing10%fetalserum to fifth generation becoming dedifferentiation chondrocytes,CCK-8wasadded after cultured1D,2D,3D,4D,5D,6D,7D.Four hours later the absorbancevalue were detected and recorded by a microplate.According to the absorbancevalue of each hole, drawing the fifth generation of chondrocyte growthconcentration curve.3.Serum preparation system with different drugsCalculate the equivalent dosage of glucosamine hydrochloride\shaoyangshenggu formular based on surface area. After the drug in vivo drugconcentrations to stabilize,the blood was centrifuged to obtain serumcontaining.4.Different drugs at different concentrations of serum containing affectchondrocyte dedifferentiation intervention.The dedifferentiated chondrocytes were randomized and cultured withserum containing different drugs at different concentrations.Observe themorphology and growth of chondrocyte under an inverted microscope. Find outthe different of proliferation and growth after different serotypes intervention.To understand the collagen type Ⅱexpression rate(%) by collagen type Ⅱstaining.Results:1.Using mechanical separation and enzymatic digestion method toestablish chondrocytes in vitro culture system is in line with chondrocyte characteristics after observation under the inverted microscope andidentification of toluidine blue staining and collagen type Ⅱ staining.Thebiological characteristics, morphology, staining characteristics of fifthgeneration of chondrocytes is in line with differentiation chondrocyte.2.The morphology of dedifferentiation of chondrocytes: dedifferentiatedchondrocyte cell morphology irregular fusiform.Loss activity, cellmembrane folds, smaller nuclei.Dedifferentiated chondrocytes for cell cycleobserved Show: dedifferentiated chondrocyte growth recovery will be1-2days after adherent,during this period, the proliferation of cell state are at astandstill.In the next2-5days, the cells have a high-speed growth period, theactivity and proliferation ability of cell is in the best condition.In the next fivedays, proliferation rate/morphology and cell viability was reduced or evendeath.3.Different concentrations of drug serum affect the fifth generation ofchondrocytes Show:The group containing glucosamine and“shaoyangshenggu formular” compared with the control group shows: theexperiment groups have more cytoplasm and larger nucleus and betterfeatures of cells,but the two experiment group show no significant differencesin cell morphology,phenotype etc. Cell proliferation was measured by CCK-8assay shows:The experiment group have a significant promotion inproliferation of dedifferentiation chondrocytes after48hours of interventionof drug serum compared with the control group(P <0.05).But in the first24 hours of intervention and after intervention72hours, containing serum ondedifferentiated chondrocyte proliferation division less (P>0.05).Thestaining of collagen type Ⅱshows after the72hours intervention:Theexperiment group have a better positive rate of collagen type Ⅱ stainingcompared with the control group(P <0.05). But the two experiment groupshow no significant differences(P＞0.05).Conclusion:1.Different concentrations of Shaoyang Shenggu formular serum canimprove the proliferation of dedifferentiation chondricytes and expression ofcollagen type II. It also helps to maintain the chondrocyte phenotype, but itsrole is limited.