| Objectives:High-level interleukin-lb (IL-1β) expression of disc cells is believed to play an important role in intervertebral disc degeneration (IVDD). Recent developments in mesenchymal stem cells (MSCs) research have shown great promise for the treatment of IVDD, BMSCs can have phenotypic differentiation of nucleus pulposus cells and restore vertebral body height in vivo. The purpose of the present study was to establish an in vitro model for bone marrow-derived mesenchymal stem cells (BMSCs) therapy by investigating the protective and therapeutic effects of BMSCs on nucleus pulposus (NP) cells.Methods:1.The experimental cell isolation and identification as well as the experimental group. We receive the NP cells and BMSCs from SD rat, NP cells were identified by collagen type Ⅱ and aggrecan immunohistochemical identification. The third generations of BMSCs phenotypes were identified by flow cytometry instrument. Experiment is divided into three groups:1. The control group:under the condition of serum free medium for 24 h and replacement of contain a complete medium for 24 h.2. Inflammatory intervention group (group A):under the condition of serum free medium to join IL-1 beta (20 ng/ml) intervention in 24 h whereafter the replacement for containing complete medium for 24 h.3. The trained group (group B):under the condition of serum, free medium to join IL-1 beta (20 ng/ml) intervention after 24 h, take into the BMSCs cells transwell Chambers with complete medium for 24 h, three groups of experiments are divided into two stages, each time for 24 h.2. The Realtime-PCR:genes expression of NP cell in each groups. The rel ative genes expression of ADAMTS-4,5,7, MMP-3,13, and TIMP-1 were detected by Realtime-PCR.3. The apoptosis rate of NP cell in each groups were detected by of the Caspase 3 activity detection kit instructions frome beyotime. And TUNEL staining for three groups of cells, observe the activity of TUNEL staining of three groups of cells. BMSCs non-contact trained under NP inflammatory intervention apoptosis percentage changes. Using the same number of GFP-BMSCs add NP cells directly as group C, The changes of apoptosis in each group were examined by the Annexin V (PE)/PI kits.4.Cell migration test:for 0 ng/ml,10 ng/ml,20 ng/ml,50 ng/ml concentration of IL 1 beta intervention of nucleus pulposus cells for 24 h, and replace transwell board to with 8-um diameter of filter plate, through the comparison of four groups after 24h, BMSCs migration ability for NP cell were observed.5.Mitochondrial staining observations:After using IL-1β(20 ng/ml) induced NP cells for 24h. Dyed by mitochondrial dye, GFP-BMSCs were take into NP cell for 24h direct-coculture, and whereafter cells slides were moved in a confocal microscope.Results:1.The Realtime PCR results:Compared with the control group, the expression of ADAMTS-4/5/7,MMP-13/3 were significant increased (p<0.05),Compared with group A, The degeneration index were significantly reduce in Group B (p<0.05). protective factor like TIMP-1 was crosscurrent.2. TUNEL staining:tTUNEL staining allows for the detection and quantification of apoptosis at the cellular level. The number of TUNEL-positive cells increased with inflammatory factors (20 ng/ml), whereas the number of TUNEL-positive cells was reduced in the co-culture group3.Detection of Caspase 3 activity:The results of the caspase-3 activity assay showed that compared with the normal group, the caspase-3 activities in the 10 ng/mL、20 ng/mL and 50 ng/mL inflammatory factor treatment groups were significantly increased. In each indirect co-culture group, the caspase-3 activity showed varying degrees of decrease. Flow cytometric analysis of annexin V-FITC(PE)/PI staining showed that apoptosis was significantly elevated in NP cells stimulated with inflammatory factors. Moreover, the apoptosis rate increased with increasing concentrations of inflammatory factors. These results indicated that co-culturing NP cells with BMSCs significantly reduced the apoptotic rate in different levels of IL-1β. And direct co-culture may have better anti-apoptosis effects than indirect co-culture. Interesting, the apoptosis rate of added BMSCs was very low.4. The number of cells migrated through pores was counted under 10 random high power fields for each group and then been compared. Amongst all the groups, the difference between negative control and IL-1β pre-induced groups was statistically significant. Cell damages caused by IL-1β enhanced the migratory ability of BMSCs in a dose-dependent manner.5.Transfer mitochondria in direct co-culture of NP Cells and BMSCs. The arrows show TnT-like structures were observed from BMSCs to NP cells by confocal microscopy. Mitochondrion was clearly seen in red fluorescence channel, transferring from relabeled GFP BMSCs to stimulated NP cells by TnTs.Conclusion:Through paracrine manner, the anti-apoptosis effects as well as anti-inflammatory effects related to BMSCs treatments in IVDD were firstly investigated in vitro in this study. BMSCs migration and integration of mitochondria to IL1-b induced NP cells were also examined, The "treatment" effects of intervertebral disc inflammatory environment cannot be ignored. The interaction between stimulated NP cells and BMSCs is conducive to simulating the in vivo process of stem cell-mediated repair. |
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