Experimental Study On Sifting And Ossification Of Bone Marrow Mesenchyal Stem Cells Of Rabbit In Vitro

Objective:1.To invest the bone marrow mesenchymal stem cells(BMSCs) of rabbit in vitro and to establish a effective method to separateand culture BMSCs in vitro.2. To make sure which is the ideal seed cellsfor experiment in tissue engineering, test the proliferation cycle and cellsgrowth cycle of BMSCs of the first,5th，10th generation.3. To identifyossification of BMSCs the osteoblast specific indicators typeⅠcollagen,alkaline phosphates and calcium nodules were detected.Methods:1. The bone marrow liquid was extracted at the femoral ofthe rabbit. The BMSCs was separated and cultured by density gradientcentrifugation and adherence method and observed in vitro.2. Observedthe BMSCs of the first,5th,10th generation, tested the proliferation cycleand cells growth cycle of BMSCs in order to understand the biologicalcharacter of BMSCs.3. BMSCs was induced by osteogenesis inducedfluid (complete medium to add dexamethasone8mol/L, betaphosphoric acid glycerol sodium10mmol/L, vitamin C50ug/ml), typeⅠcollagen, alkaline phosphates (ALP), calcium nodules were respectivelydetected at the7th day，14th day and the21st day in order to research the osteoblast differentiation ability of BMSCs.Result:1. Active BMSCs can be isolated by density gradientcentrifugation and adherence method. After cultured at24hours a smallamount of cells were long shuttle and mostly were short spindle. At the72hours the adherent cells increased significantly, and most cells werelong spindle. On the5th days, cell proliferated greatly and severalobviously cell masses were formed. On the7th days the cells achievedfusion.2. The first,5th,10th generation cells were tested by MTT andcell flow cytometry, proliferation ability and growth cycle of the firstgeneration and the5th generation of has no significant difference (P>0.05), proliferation ability and cell growth cycle of the10th generationwas obvious different from the5th and first generation cells (P <0.05).3Ossification different of BMSCs: along with the time went on, the size ofthe cells became polygon. Induced group of cells present contactinhibition, normal group present overlaps growth. TypeⅠ collagen wasactive in the induced group, in normal group typeⅠ collagen wasinactive. ALP enzyme linked immunosorbent assay show that inducedgroup had a significant difference with normal group c (P <0.05).Induced group calcium nodules were red dyed, but the normal group wasinactive. These results show the5th generation BMSCs in certainconditions can be induced to the osteoblast and its ossification is strong.Conclusion:1. Density gradient centrifugation and adherencemethod can isolate higher purity and activity of BMSCs.2. Through thegeneration of BMSCs morphological observation, cell proliferation cycleand cell growth cycle test, confirmed that each generation of first,5thbetween cells of biological characteristics of no significant difference,10 th generation cells had obvious difference with first,5th generation cells.The5th generation is the only cell line. It is claimed that the fifthgeneration cells are ideal seed cells for tissue engineering.3.TypeⅠcollagen, ALP，calcium nodules detection results showed that the5thBMSCs in specific conditions can be induced into the osteoblast and itsossification is strong...