Wang Yuntao, Wu Xiaotao, Wang Feng, Hong Xin, Bao Junping, Zhu Lei
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2014 Jun;28(6):758-62.
To investigate the biological characteristics of bone marrow mesenchymal stem cells (BMSCs) in microenvironment of premature senescence of nucleus pulposus cells (NPCs) so as to lay a foundation for the repair of intervertebral disc degeneration by BMSCs transplantation.
Human degenerative nucleus pulposus and normal bone marrow were collected, and then NPCs and BMSCs were isolated, cultured, and identified. The 3rd passage BMSCs and the 1st passage NPCs with premature senescence were co-cultured without contact in the Transwell culture system. NPCs to BMSCs ratio was 75%:25% (group A), 50%:50% (group B), and 0:100% (group C). The morphological changes of BMSCs were observed by inverted phase contrast microscopy and transmission electron microscopy. At 3 and 6 days after co-culture, cell counting kit 8 was used to detect cell viability, flow cytometry was used to observe the cell cycle and detect DNA metabolism after BrdU labeling. Cell senescence was also evaluated by detecting senescence associated β-galactosidase (SA-β- gal) activity.
The typical morphology of cell senescence was seen in groups A and B, especially in group A. At 3 and 6 days after co-culture, the cell survival rate of group A was significantly lower than that of group B (P < 0.05). At 3 days after co-culture, the proportion of cells in G1 phase in group A was significantly higher than that in groups B and C (P < 0.05), the proportion of cells in S phase in group A was significantly lower than that in groups B and C (P < 0.05). At 6 days, the proportion of cells in G1 phase in group A was about 81.0%, and the proportion of cells in S phase and G2 phase decreased, showing significant difference when compared with groups B and C (P < 0.05); the proportion of cells in G phase in group B was about 74.4%, showing significant difference when compared with group C (P < 0.05). BrdU content in group A was significantly lower than that in groups B and C at 3 and 6 days after co-culture (P < 0.05), but no significant difference was found between groups B and C at 3 days (P > 0.05); BrdU content in group B was also significantly reduced when compared with group C (P < 0.05) at 6 days. At 6 days, SA-β-gal activity was significantly increased in groups A and B, and significant difference was shown in SA-β-gal positive cell number between groups (P < 0.05).
Premature senescence of NPCs can down-regulate the proliferation capacity of co-cultured BMSCs by the paracrine effect. The greater proportion of NPCs with premature senescence is, the earlier senescence of BMSCs will be induced.
研究髓核细胞（NPCs）早衰微环境中骨髓间充质干细胞（BMSCs）的生物学特性，为BMSCs移植修复椎间盘退变奠定基础。
收集人退变髓核与正常骨髓，分离、培养并鉴定NPCs和BMSCs。将第3代BMSCs与早衰的第1代NPCs在Transwell培养系统中进行非接触共培养。NPCs与BMSCs比例分别为75%:25%（A组）、50%:50%（B组）和0:100%（C组）。通过倒置相差显微镜和透射电子显微镜观察BMSCs的形态变化。共培养3天和6天后，使用细胞计数试剂盒8检测细胞活力，采用流式细胞术观察细胞周期并检测BrdU标记后的DNA代谢。通过检测衰老相关β-半乳糖苷酶（SA-β-gal）活性评估细胞衰老情况。
A组和B组出现典型的细胞衰老形态学变化，尤其是A组。共培养3天和6天后，A组细胞存活率显著低于B组（P<0.05）。共培养3天时，A组G1期细胞比例显著高于B组和C组（P<0.05），A组S期细胞比例显著低于B组和C组（P<0.05）。6天时，A组G1期细胞比例约为81.0%，S期和G2期细胞比例下降，与B组和C组相比差异显著（P<0.05）；B组G1期细胞比例约为74.4%，与C组相比差异显著（P<0.05）。共培养3天和6天后，A组BrdU含量显著低于B组和C组（P<0.05），但3天时B组和C组之间差异无统计学意义（P>0.05）；6天时B组BrdU含量与C组相比也显著降低（P<0.05）。6天时，A组和B组SA-β-gal活性显著升高（P＜0.05），组间SA-β-gal阳性细胞数差异显著（P<0.05）。
NPCs早衰可通过旁分泌作用下调共培养BMSCs增殖能力，早衰NPCs比例越高，诱导BMSCs衰老越早。
