Experimental Study Of Indirect Co-culture System On Differentiation Of Rabbit Bone Marrow Mesenchymal Stem Cells Into Nucleus Pulposus-like Cells

PartⅠ：Isolation、culture of rabbit bone marrow mesenchymal stem cells in vitro bydensity gradient centrifugation plus adherence method and its morphology studyObjective To explore the approaches of isolation、 culture、 identification andamplification of the rabbit bone marrow mesenchymal stem cells in vitro, to observe thegrowth and activity of the BMSCs. Methods Density gradient centrifugation and adherentculture method isolated and purified BMSCs then proliferated in vitro. The growth of primaryand passaged cell was observed under the inverted microscope, count the number of cells anddraw growth curve. HE staining was used to observe the morphologic features, and examinethe expressions of cell surface antibody of CD29、CD34and CD44by immunocytochemistry.Results BMSCs of rabbit were isolated、cultured and proliferated in vitro successfully.Growth activity was observed, The1-3generation BMSCs proliferated more rapidly andvigorously. The proliferation of BMSCs decreased while the cell proliferated moregenerations. These cultured cells showed immunoreactivity to CD29、CD44but not CD34.Conclusion Purified BMSCs of rabbit could be obtained via density gradient centrifugationcombined with adherent culture method The1-3generation with high proliferation can beused as seed cells for tissue engineering in further research work. Part Ⅱ： Different generations of rabbit nucleus pulposus cells: Morphologicalobservation and biological propertiesObjective To investigate the characteristics of different generations of rabbit nucleuspulposus cells, searching for the best suitable seed cells to treat degenerative disc diseases.Methods Nucleus pulposus cells from New Zealand white rabbits were separated, culturedand then passaged. The morphological changes of primary, passages3and4nucleus pulposuscells were observed by hematoxylin-eosin staining under an inverted microscope. The biological properties of rabbit nucleus pulposus cells were observed. Aggrecan and type Ⅱcollagen expressions were detected by toluidine blue and immunocytochemistry staining,respectively. TypeⅡ collagen and glycosaminoglycan mRNA expressions in the nucleuspulposus cells were detected by reverse transcription-PCR. Result Rabbit nucleus pulposuscells were successfully cultured and passaged. Primary NP cell is round or polygonal, theaverage adherence time is7days. The first three generations of the cultured nucleus pulposuscells were round or polygonal, have strong vitality. HE staining shows nuclei were stained auniform blue-black, cytoplasm showed light pink, the cytoplasm of nucleus pulposus cellswas sky blue, toluidine blue staining and type II collagen immunohistochemical stainingshows the cytoplasm of nucleus pulposus cells were showed yellowish-brown. The4thgeneration of NPCs had degeneration and the level of the Type Ⅱ collagen andglycosaminoglycan mRNA expression compared to previous generations was significantlydecreased. Conclusion The first three generations of nucleus pulposus cells are metabolicexuberant and phenotype consistent and normal expression of aggrecan and type II collagen,the4th generation began to age and degenerate. Part Ⅲ： Experimental study of rabbit bone marrow mesenchymal stem cellsdifferentiation into nucleus pulposus-like cells in indirect co-culture systemObjective To research the differentiation potential of rabbit bone marrowmesenchymal stem cells(BMSCs) into nucleus pulposus-like cells（NPCs)，so as to provide alarge number of cells for biotherapy of degenerative disc disease. Methods BMSCsextracted from rabbit were cultivated and went down to the third passage cells, then NPCsextracted from rabbit. the third generation BMSCs and NPCs are divided into three groups,only BMSCs were cultured, only NPCs were cultured, the third passage BMSCs wereco-cultured with NPCs, Transwell six-well plates were used for coculture without contact.Culturing cells were observed by inverted contrast phase microscope. The cells were culturedfor7d. immunocytochemistry、RT-PCR and Western-blot were used to examine the expressionof type II collagen and aggrecan, then the result of each group of cells were analyzed. Results BMSCs and NPCs were successfully isolated，the results of immunocytochemistry、RT-PCRand Western-blot showed that the expressed of type II collagen and aggrecan in the BMSCs ofco-culturing group and single culturing NPCs were similar. the expressed of type II collagenand aggrecan in the BMSCs of co-culturing group and single culturing NPCs were higher thansingle culturing BMSCs. Conclusion BMSCs have the qualities to differentiate towardNPCs, It has been found that coculture of BMSCs with NPCs can induce BMSCsdifferentiating into an NPCs phenotype，indicating that BMSCs may provide sufficientsources of cells for the biological treatment of degenerative disc disease.