Effect On Biological Characteristics Of Human Beings Nucleus Pulposus Mesenchymal Stem Cell Under Different Acid Condition

Objectives To observe the biological effects of acid condition on human nucleus pulposus mes -enchymal stem cells (NPMSCs) in vitro, and to explore the mechanism of intervertebral disc degeneration.Methods Cells were isolated from non-degenerative nucleus pulposus(Pfirrmann I or II) which were harvested from 6 patients with scolisis and then cultured and passaged in vitro. The immunophenotype of the second generation cells were firstly identified by flow cytometry, and then were induced into osteogenesis, chondroblast and adipogenic cells which were identified byalizarin red, Oil red O and toluidine blue staining, respectively. The cells were firstly identified by the criteria of the International Society for Cellular Therapry(ISCT), and then the cells were further incubated in different pH conditions (pH=6.2,6.5,6.8,7.1,7.4). The cell proliferations were detected by cell counting kit-8(CCK-8) after culturing for 1,3,5,7,9,11,13 days, using flow cytometry observe the apotosis rates after culturing for 3 days, and the mRNA expressions of genes(Jagl, Notchl et al) and the acid sensing ion channel(ASIC) genes(ASIC1, ASIC2, ASIC3, ASIC4) were detected by semi-quantitative real-time PCR after culturing for 28 days.Results All the original cells from non-degenerative nucleus pulposus could grow adhering to the wall. The immunephenotype results showed all the cells highly expressed CD90(96%), CD 105(95%), CD73(94%), and lowly expressed CD45, CD34, HLA-DR(less than 4%). And multilineage results showed cells from normal nucleus pulposus were stained red calcium salts by alizarin red, and adipocyte-like cells were stained red, and chondrocyte-like cells were dyed blue.Based on the above results, the cells obtained from non-degenerative nucleus pulposus met the criteria of ISCT, and the cells were absolutely mesenchymal stem cells. Cell proliferation assay by CCK-8 showed there was no statistic signification(P>0.05) after culturing at 1 day and 3 days, while there were obvious differences(P<0.05) between the any two groups after culturing at 5,7,9,11,13 days, and the OD value was indirectly related to pH value, the apoptosis rate was statistically significant (P<0.05) between any two pH groups, and pH 7.4 group had the lowest apoptosis rate. QRT-PCR results showed that the m RNA expression of stem cells-related genes(Oct4, Nanog, Jagl, Notch1) and ASIC genes(ASIC1, ASIC2, ASIC3, ASIC4) of pH 7.4 group was higher than that of pH 7.1 group, pH 6.8 group, pH 6.5 group, and pH 6.2 group. With the pH value of culture medium decreased, the cell apotosis rate increased, while the mRNA expression of stem cells-related genes and ASIC genes decreased.Conclusions Low acid environment has no effect on the proliferation of NPMSCs after culturing at 1 and 3 days, while has obvious effect on the proliferation and m RNA expression of NPMSCs and promotes the apoptosis of NPMSCs after culturing 5 days, and the effect increases with the pH value decreases.