Comparative study of the biological effects from two kinds of human stromal stem cells to degenerative nucleus pulposus cells under the condition of contact co-culture

English Keywords:Contact co-culture  Mesenchymal stem cells  Degenerative nucleus pulposus cells  Comparative study

English Abstract:

  【Abstract】 Objectives: To compare the biological effects of degenerative nucleus pulposus cells when co-cultured with two kinds of people’s mesenchymal stem cells under a contact system. Methods: The degenerative nucleus pulposus tissue, dipose tissue and bone marrow were obtained from the same patient who suffered from lumbar disc herniation during the surgery, and then degenerative nucleus pulposus cells(NPCs), adipose- derived mesenchymal stem cells(ADSCs) and bone marrow mesenchymal stem cells(BMSCs) were isolated and cultured. The third generation of cells had the direct cell-cell contact co-culture. First, the third generation of degeneration NPCs was labled by PKH26, and then the NPCs with ADSCs and BMSCs were put in 6-well plates to contact co-culture at the ratio of 1∶1. There were three groups: group A was NPCs cultured alone, as the control group; group B was NPCs co-cultured with ADSCs; group C was NPCs co-cultured with BMSCs. On the seventh day after co-culture, BMSCs, ADSCs and NPCs were seperated by the co-culture flow cytometry, the total RNA of each group was extracted, then the Real-Time PCR technology was used to detect the relative gene expression of type Ⅱ collagen, proteoglycan and SOX-9. The results were statistical analyzed by t test between every two groups, P<0.05 meant statistically significant. Results: After obtained the degenerative nucleus pulposus tissue, fat tissue and bone marrow tissue, the NPCs, ADSCs and BMSCs were successfully isolated and cultured. When cells were transmitted to the third generation, the NPCs labled by PKH26 were got successfully. On the seventh day after co-culture, MoFlo high-speed flow cytometry was used to separate and obtain the NPCs in group B and group C. Total RNA was extracted, after reversing transcription and Real-time PCR, the collagen protein Ⅱ, proteoglycan and SOX-9 gene relative expression of NPCs in each group were obtained. Collagen protein Ⅱ, proteoglycan and SOX-9 of NPCs in group A was 1.03±0.04, 1.05±0.07, 1.04±0.17; 5.26±0.24, 7.71±0.21, 3.84±0.25 respectively in group B; and 9.33±0.39, 11.07±0.34, 5.64±0.26 respectively in group C. Compared with group A, the nucleus pulposus cells of group B and C had a significant increase of collagen protein Ⅱ, proteoglycan and SOX-9 gene expression(P<0.05); The group C had a higher significant increase of gene expression of collagen protein Ⅱ, proteoglycan and SOX-9 than group B(P<0.05). Conclusions: BMSCs and ADSCs can both stimulate and activate the degenerative NPCs under the condition of contact co-culture. Compared with ADSCs, BMSCs have a stronger activation effect on degenerative NPCs, it may be more suitable for degenerative disc diseases′ biological treatment.