| Background and Purpose: Degenerative disc disease (disc degeneration disease,DDD) is one of the serious problems of human medical problems, and the incidenceincreased year by year. Although traditional surgical methods achieved good effect, but itfailed to resolve the nature of disc degeneration fundamentally. With the advances inbiological therapy, and the stem cell technology continues to evolve and tissue engineeringtechniques, people has brought new prospects in the treatment of degenerative disc disease.In these studies, the choice of the seed cells, is an important aspect of treatment decisions.Currently, more in-depth study is Bone mesenchymal stem cells (BMSCs) andadipose-derived stem cells (ADSCs), which were primitive stromal population isolated from bonemarrow and adipose tissue, which were researcheddeeply currently, both of them had thebasic characteristics of the stem cell, such as growed as adherent cells, self-renewing, andexpressed cell surface markers displayed by MSCs, what’s more, could be differentiatedinto other cells. In this study, we extracted the bone marrow, adipose tissue and thedegenerative nucleus pulposus tissue from the same patient. BMSCs, the ADSCs and thedegenerative nucleus pulposus cells (NPCs) were isolated from fresh samples, thenco-culture the BMSCs and ADSCs with degenerative NPCs in six-well plates under thecontact condition. We use the MoFlo high speed flow cytometry to separate thedegenerative NPCs co-cultured with the BMSCs and ADSCs, then the Real-Time PCRtechnology was used to detect the relative gene expression of the typeⅡcollagen,proteoglycan and SOX-9.To investigation the biological effects of degenerativenucleus pulposus cells when co-cultured with BMSCs and ADSCs under a contact system,and compare the difference of the biological effects between the BMSCs and ADSCs. Withthe purpose of provide the more suitable stem cells to treat the disc degeneration disease(DDD).Methods: Obtained the degenerative nucleus pulposus tissue, dipose tissue and bone marrow from the same patient who suffered lumbar disc herniation during the surgery, andthen isolated and cultured degenerative nucleus pulposus cells (NPCs), adipose-derivedmesenchymal stem cells (ADSCs) and bone marrow mesenchymal stem cells (BMSCs).Used the third generation of cells for the direct cell-cell contact co-culture.First, we labledthe third generation degeneration NPCs by PKH26, and then put the NPCs with ADSCsand BMSCs in6-well plates to contact co-culture at the ratio of1:1. There were threegroups: Group A was NPCs cultured alone, as the control group; Group B was NPCsco-cultured with ADSCs; Group C was NPCs co-cultured with BMSCs. After7days ofco-culture, separated of BMSCs, ADSCs and NPCs by the co-culture flow cytometry，thetotal RNA of each group were extracted, then the Real-Time PCR technology were used todetect the relative gene expression of typeⅡcollagen, proteoglycan and SOX-9. The resultswere statistical analysis by t test between groups, P <0.05means statistically significant.Results: After we obtained the degenerative nucleus pulposus tissue, fat tissue andbone marrow tissue, we successfully isolated and cultured the NPCs, ADSCs and BMSCs.When cells were transmitted to the third generation, we got the NPCs labled by PKH26successfully. After7days of co-cultured, we used MoFlo high-speed flow cytometry toseparate and obtain the NPCs in Group B and Group C. Total RNA was extracted, afterreverse transcription and Real-time PCR, we obtained the Ⅱ collagen protein,proteoglycan and SOX-9gene relative expression of NPCs in each group. Ⅱ collagenprotein, proteoglycan and SOX-9of NPCs in Group A were1.03±0.04,1.05±0.07,1.04±0.17;5.26±0.24,7.71±0.21,3.84±0.25in Group B were; and9.33±0.39,11.07±0.34,5.64±0.26in Group C. After statistical analied by Student t test between groups, wefound that compared with Group A, the nucleus pulposus cells of group B and C had asignificant increase of Ⅱ collagen protein, proteoglycan and SOX-9gene expression (P <0.05); The group C had a more significantly increased with gene expression of Ⅱcollagen protein, proteoglycan and SOX–9than group B,(P <0.05).Conclusions:Bone marrow mesenchymal stem cells and adipose mesenchymal stemcells can both stimulate and activate the degenerative nucleus pulposus cells under thecondition of contact co-culture; Compared with adipose mesenchymal stem cells, Bonemarrow mesenchymal stem cells have a stronger activation effect for degenerative nucleuspulposus cells, itmay be more suitable for degenerative disc diseases’ biological treatment. |
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