The Comparative Experimental Research Of The Culture Of Rabbit Bone Marrow Mesenchymal Stem Cells By Different Methods

Objective: To establish the method of culture of the rabbit bone marrow mesenchymal stem cells in vitro.It also compares the method of density gradient centrifugation with the directly adherent method.Using bone marrow mesenchymal stem cells and amniotic membrane to construct biomaterial for conjuctiva defect.Methods:1 Extract marrow from 3 months male New Zealand white rabbits in sterile condition.Isolate and culture the rabbit bone marrow mesenchymal stem cells by the methods of density gradient centrifugation and the directly adherent.Continous passage culture and observe the cells.2 Culture the third passage of the bone marrow mesenchymal cells on the culture plate which have cover glass in it.When the percents of the cells reach about 90% on the glass,taking it out and doing the reaserch of the immunohistochemical staining.The make a compare about the results of the immunohistochemical of the two different methods.3 Inoculate the third passage of the bone marrow mesenchymal cells in good condition to the basal surface of the amniotic membrane.When the cells mix together,taking a picece of amniotic membrane and observing it with scanning electron microscope.Results:1 The bone marrow mesenchymal cells cultured by different methods have the same morphology.They are all areatus.They present spindle and polygon shapes.The sub-culture cells are also uniform.They proliferate actively and have short growth cycle.2 The immunohistochemical staining display that the antibody of CD44 is positive and the antibody of the CD34 is negative.3 The mean optical density of CD44 by the method of the directly adherent method is lower than the method of density gradient centrifugation.The result of CD34 is opposite.4 The bone marrow mesenchymal stem cells can grow on the surface of the amniotic membrane and mix well with it in the scanning electron microscope.Conclusion:1 The bone marrow mesenchymal stem cells of the rabbit can be cultured and the directly adherent.They all can be passaged and the state of the cells is well.2 The results of the immunohistochemical support the cells are bone marrow meshenchymal cells.3 The method of the density gradient centrifugation obtain cell purity is better than the method of the the whole bone marrow adherent.4 The amniotic membrane can construct the biomaterial as the carrier material of the bone marrow mesenchymal stem cells.