The repair effects of ISN-SCs on senescence nucleus pulposus cells

English Keywords:Intervertebral disc degeneration  Nucleus pulposus cell  Senescence  Repair

English Abstract:

  【Abstract】 Objectives: To investigate the repair effects of ISN-SCs(stem cells derived from stem cell niches of intervertebral disc) on senescence NPCs(nucleus pulposus cells). Methods: 10 Sprague-Dawley rats(male, 10-week-old) were used for this study. The functional spinal units were isolated after the animals being sacrificed. Then, the tissues of ISN (stem cell niches of the intervertebral disc) and nucleus pulposus were isolated anatomically under dissecting microscope. After being digested and filtrated, the harvested primary ISN-SCs and NPCs were resuspended, seeded and cultured under normal condition. The cells were passaged, and P4 ISN-SCs and P3 NPCs were used for further experiments. P4 ISN-SCs were cultured in specific differentiation induction medium (osteogenesis, chondrogenesis and adipogenesis) for 1 induction period before being tested. Alizarin red staining and calcium cobalt staining were used for detecting osteogenesis, alcian blue staining was used for detecting chondrogenesis, and Oil red O staining was used for detecting adipogenesis. The senescence model of NPCs was established by continuous passage, and the effectiveness of model was detected by the staining of senescence associated-β-galactosidase(SA-β-Gal). The cell culture systems were divided into 3 groups: normal control group, senescence group, and co-culture group. Treatments were set as follows P3 NPCs were seeded in the normal control group, senescence NPCs were seeded in the senescence group, and in the co-culture group, P4 ISN-SCs and senescence NPCs were mixed at the blending ratio 1:1 and seeded, total cell number and the culture environment of each specimen were the same. After being cultured for 7 days, further detections were conducted in each group, including toluidine blue staining for aggrecan, immumohistochemical staining for type Ⅱ collagen, and quantitative real-time polymerase chain reaction(qPCR) for the mRNA expression level of ACAN and COL2a1, which were compared among groups. Results: Osteogenic, chondrogenic and adipogenic differentiation of ISN-SCs were observed under the certain induction environment respectively. SA-β-Gal staining indicated that NPCs gradually became senescence as passage increased, with no obvious senescence cells in passage 3(3.0±0.5)%, less than 20% senescence cells in passage 5(18.3±0.7)%, and more than 80% senescence cells in passage 10 (86.0±4.6)%. When compared to the normal control group, the senescence group expressed less aggrecan and type Ⅱ collagen (quantification, P<0.05). While after co-culturing, the co-culture group expressed more aggrecan and type Ⅱ collagen (quantification, P<0.05) when compared to the senescence group, which was comparable to the normal control group. QPCR results showed that the mRNA expression levels of ACAN and COL2a1 in senescence group significantly decreased when compared to the normal control group (1.00±0.05 vs 0.43±0.03, P<0.05 and 1.00±0.03 vs 0.40±0.02, P<0.05, respectively), and that the mRNA expression levels of ACAN and COL2a1 in co-culture group were significantly increased when compared to the senescence group (0.43±0.03 vs 0.99±0.05, P<0.05 and 0.40±0.02 vs 1.07±0.04, P<0.05, respectively), which was comparable to the normal control group. Conclusions: ISN-SCs possess excellent multilineage differentiation capacities. The senescence NPCs can be remarkably repaired in co-culture model, through elevating the expression levels of extra cellular matrix protein and mRNA. ISN-SCs can be utilized for further research on the repair of intervertebral disc degeneration.