Effect on biological characteristics of human beings nucleus pulposus mesenchymal stem cell under different acid condition

English Keywords:Intervertebral disc degeneration  Nucleus mesenchymal stem cell  Acid environment  Biological characteristics

English Abstract:

  【Abstract】 Objectives: To observe the biological effects of acid condition on human nucleus pulposus mesenchymal 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(CD105, CD90, CD73, CD45, CD34 and HLA-DR) of the second generation cells were firstly identified by flow cytometry, and then were induced into osteogenesis, chondroblast and adipogenic cells which were identified by alizarin 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 cultured in different acid conditions(pH=6.2, 6.5, 6.8, 7.1, 7.4, respectively). The cell proliferations were detected by cell counting kit-8(CCK-8) after culturing for 1, 3, 5, 7, 9, 11, 13 days, the cell apotosis rates were observed by flow cytometry after culturing for 3 days, and the mRNA expressions of stem cells-related genes(Oct4, Nanog, Jag1, Notch1) 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%), CD105(95%), CD73(94%), and lowly expressed CD45, CD34, HLA-DR(all less than 4%). Multilineage differentiation results showed cells obtained from normal NP developed red stained calcium salts, and adipocyte-like cells which were stained red by Oil red O, and chondrocyte-like cells were stained blue by toluidine blue. According to the above results, the cells obtained from non-degenerative nucleus pulposus met the criteria of ISCT, and the cells were definitely 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. According to the cell apoptosis rates, there were statistic significations(P<0.05) between any two pH groups, and pH 7.4 group had the lowest apoptosis rate. QRT-PCR results showed that the mRNA expression of stem cells-related genes(Oct4, Nanog, Jag1, 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 mRNA expression of NPMSCs and promotes the apoptosis of NPMSCs after culturing 5 days, and the effect increases with the pH value decreases.