论文部分内容阅读
目的探讨在过早衰老髓核细胞(nucleus pulposus cells,NPCs)微环境中BMSCs的生物学行为,为充分利用BMSCs干细胞特性进行退变椎间盘修复奠定实验基础。方法收集人退变髓核组织与正常骨髓组织进行NPCs、BMSCs分离、培养及鉴定;对过早衰老诱导的第1代NPCs和正常第3代BMSCs行非接触共培养,根据NPCs与BMSCs不同比例将实验分为A组(75%∶25%)、B组(50%∶50%)和C组(0∶100%)。倒置相差显微镜和透射电镜观察各组BMSCs形态变化;对培养3、6 d的BMSCs进行增殖能力检测:细胞计数试剂盒8检测细胞活力、流式细胞仪检测细胞周期、BrdU标记流式检测DNA代谢;培养6 d检测衰老相关β半乳糖苷酶(senescence associatedβ-galactosidase,SA-β-gal)活性评价细胞衰老。结果倒置相差显微镜示A、B组BMSCs中混杂的三角形或大多角形细胞增多,尤其是A组;透射电镜观察A、B组均可见典型衰老形态的细胞。培养3、6 d,A组细胞存活率均显著低于B组,差异有统计学意义(P<0.05)。3 d时,A组G1期细胞比例明显高于B、C组(P<0.05),S期细胞比例明显低于B、C组(P<0.05)。6 d时,A组约81.0%细胞停滞于G1期,S期及G2期细胞比例减小,与B、C组比较各细胞周期分布差异均有统计学意义(P<0.05);B组细胞停滞于G1期比例增加至约74.4%,与C组比较各细胞周期分布差异均有统计学意义(P<0.05)。培养3、6 d,A组掺入的BrdU含量均明显低于B、C组(P<0.05);B、C组间3 d时差异无统计学意义(P>0.05),6 d时B组明显低于C组(P<0.05)。培养6 d,A、B组SA-β-gal活性显著提高,3组间比较差异均有统计学意义(P<0.05)。结论过早衰老的NPCs可通过旁分泌效应下调共培养的BMSCs增殖能力,而NPCs数量上的优势使得共培养的BMSCs更早表现出衰老特征。
OBJECTIVE: To investigate the biological behavior of BMSCs in prematurely aged nucleus pulposus cells (NPCs) microenvironment, and lay the experimental foundation for making full use of the characteristics of stem cells of BMSCs for degenerative disc repair. Methods NPC degeneration and normal bone marrow tissue were collected for NPCs, BMSCs isolation, culture and identification; premature aging-induced first-generation NPCs and normal third-generation BMSCs were non-contact co-culture, according to NPCs and BMSCs at different ratios Experiments were divided into group A (75%: 25%), group B (50%: 50%) and group C (0: 100%). The morphological changes of BMSCs in each group were observed by inverted phase contrast microscope and transmission electron microscopy. Proliferation of BMSCs cultured for 3 and 6 d were measured. Cell counting kit 8 was used to detect cell viability. Flow cytometry was used to detect cell cycle. BrdU labeling flow cytometry The senescence-associated β-galactosidase (SA-β-gal) activity was detected on the 6th day to evaluate the cell senescence. Results Inverted phase contrast microscope showed that the number of mixed triangular or large polygonal cells in group A and group B was increased, especially in group A. Transmission electron microscopy showed typical aging cells in group A and B. After 3 and 6 d of culture, the cell viability in group A was significantly lower than that in group B, with a significant difference (P <0.05). At 3 days, 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 was significantly lower than that in groups B and C (P <0.05). At 6 days, about 81.0% of cells in group A were arrested in G1 phase and the proportion of cells in S phase and G2 phase was decreased. There were significant differences in cell cycle distribution between groups B and C (P <0.05) The proportion of arrested cells in G1 phase increased to about 74.4%. The differences in cell cycle distribution between the two groups were statistically significant (P <0.05). BrdU incorporation in group A was significantly lower than that in group B and C at 3 and 6 d (P <0.05). There was no significant difference at 3 d between group B and C (P> 0.05) Group was significantly lower than the C group (P <0.05). After 6 d of culture, the activity of SA-β-gal in group A and group B increased significantly, with significant difference between the three groups (P <0.05). Conclusion Premature aging NPCs can down-regulate the proliferation ability of co-cultured BMSCs through paracrine effect, while the quantitative advantages of NPCs make co-cultured BMSCs exhibit earlier aging characteristics.