Experimental Study Of Rabbit BMSCs Differentiation To Nucleus Pulposus-like Cells Induced By NPCs Under A Variety Of Conditions

Objective To establish the non-contact co-culture system of BMSCs and NPCs in vitro, and to explore the effect and influencing factors of BMSCs differentiation to nucleus pulposus-like cells induced by NPCs under a variety of conditions.Methods 1. Four New Zealand rabbits were taken and BMSCs were obtained by using the modified whole bone marrow adherent method. BMSCs were cultured, purified and amplified in vitro.2. Biological characteristics of BMSCs were observed and identified through cell morphological observation, cell growth curve, and FCM examination, etc.3. Four New Zealand rabbits were taken and NPCs were obtained by using a modified enzyme-linked digestion method. Primary and subculture of NPCs were performed using the technology of cell culture in vitro.4. Through the cell morphological observation, the cell growth curve, toluidine blue staining, immunohistochemical staining, and RT-PCR detection, biological characteristics of NPCs were observed and identified.5. According to the different proportion of cells taken, cultured and well-identified BMSCs and NPCs were divided into different groups, namely, co-culture group (2:1), co-culture group (1:1), co-culture group (1:2), co-culture group (1:4), co-culture (1:1)+TGF-β1 group, and TGF-β1-induced group as a control. The BMSCs and NPCs non-contact co-culture system were then established. After 15 days of cultivation, real-time fluorescence quantitative PCR was used to detect the BMSCs inducted for each group. The induction effects were observed at the genetic level through the measurement of the relative expression of Aggrecan and Collagen Ⅱ genes and the results were analyzed.Results 1. BMSCs could be obtained by using the modified whole bone marrow adherent method, with a high purity confirmed by FCM.2. NPCs could be obtained by using a modified enzyme-linked digestion method, which showed expressions of Aggrecan and Collagen Ⅱ identified at both genetic and protein levels.3. In different conditions, both the NPCs and BMSCs non-contact co-culture system and the TGF-β1 induction system could induce the differentiation of BMSCs into nucleus pulposus-like cells, and positive expressions of Aggrecan and Collagen genes were revealed by real-time fluorescence quantitative PCR detection.4. In co-culture systems with different proportion of cells, those with a higher proportion of NPCs showed higher level of Aggrecan and Collagen Ⅱ gene expression in induced nucleus pulposus-like cells than those systems with lower NPds. There were significance in statistics.5. In co-culture conditions, the Aggrecan gene expression level of nucleus pulposus-like cells induced in the co-culture (1:1)+ 10ng/ml TGF-β1 group was higher than that of the co-culture group (1:4), with a statistically significant difference. Collagen II gene expression levels in both co-culture groups were close. There were no significance in statistics.6. After BMSCs differentiation to nucleus pulposus-like cells were induced by TGF-β1 in vitro, the Aggrecan and Collagen Ⅱ gene expression levels of induced nucleus pulposus-like cells were close to that of the co-culture group (1:2). There were no significance in statistics.Conclusion:NPCs could induce the BMSCs differentiation into nucleus pulposus-like cells in both conditions of TGF-β1 induction and co-culture. Both the ratio of BMSCs/NPCs and content of TGF-β1 in the systems played an important role in the induction effect.