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目的:观察酸环境对体外培养的成人髓核间充质干细胞(NPMSCs)生物学特征的影响,探讨椎间盘退变的可能机制。方法:用胶原酶消化法从6例脊柱侧凸矫形患者手术摘除的6个腰椎间盘(Pfirrmann椎间盘退变分级为Ⅰ级或Ⅱ级)髓核组织中分离细胞,体外培养,传代并观察细胞形态。取P2代细胞,利用流式细胞仪对分离得到的细胞表型CD34、CD45、CD73、CD90、CD105和人类白细胞抗原(HLA)-DR表达情况进行检测;用成骨、成软骨、成脂培养液诱导培养细胞,2周后分别用茜素红、甲苯胺蓝、油红O对细胞进行染色,观察其成脂、成骨、成软骨能力。按照国际干细胞治疗协会(ISCT)有关间充质干细胞(MSCs)的判定标准,对分离得到的细胞进行综合评估鉴定。在37℃、21%O2、5%CO2的细胞培养箱中用不同p H值(6.2、6.5、6.8、7.1、7.4)的DMEM10%血清培养液培养P2代细胞,1、3、5、7、9、11、13d后利用细胞增殖试剂盒(cell counting kit-8,CCK-8)检测细胞增殖活力(OD值),培养3d时借助流式细胞仪检测细胞凋亡率,不同p H值培养基培养28d时采用实时荧光定量(RT-PCR)检测P2代细胞“干性维持”基因Oct4、Nanog、Jag1、Notch1及酸离子通道家族蛋白基因ASIC1、ASIC2、ASIC3、ASIC4的m RNA表达情况。结果:P0代细胞均贴壁生长。P2代细胞免疫表型鉴定显示MSCs表面分子标记CD90、CD105、CD73表达比例分别高达96%、95%、94%以上,低表达CD45、CD34、HLA-DR(均低于4%)。茜素红染色、油红O染色及甲苯胺蓝染色分别证实分离的细胞均可向骨、脂肪及软骨细胞三系诱导分化。按照ISCT有关MSCs的判定标准,分离得到的细胞即NPMSCs。细胞代谢活性测定示P2代细胞在培养后1、3d各组间细胞OD值差异无统计学意义(P>0.05);5d、7d、9d、11d、13d各组间细胞OD值差异有统计学意义(P<0.05),且p H值越低细胞的OD值越小。p H值7.4组的细胞凋亡率均小于其余各组,各组间有统计学差异(P<0.05)。p H值7.4组“干性维持”基因Oct4、Nanog、Jag1、Notch1及酸离子通道家族蛋白基因ASIC1、ASIC2、ASIC3、ASIC4的m RNA均明显高于p H值7.1、6.8、6.5、6.2组(P<0.05)。随着培养液p H值降低,细胞的凋亡率升高,细胞干性维持基因及酸通道家族蛋白基因的m RNA表达降低。结论:低p H值的酸环境培养1、3d对成人NPMSCs增殖无明显影响,培养5d后明显抑制NPMSCs的增殖和基因表达,促进细胞凋亡,且随着p H降低作用越明显。
Objective: To observe the effect of acid environment on the biological characteristics of cultured human mesenchymal stem cells (NPMSCs) cultured in vitro and to explore the possible mechanism of degeneration of intervertebral disc. Methods: Six lumbar intervertebral discs (Pfirrmann disc herniation grade Ⅰ or Ⅱ) were excised from 6 cases of scoliosis orthopedic surgery by collagenase digestion. The cells were cultured, passaged and observed for cell morphology . The P2 generation cells were collected and the cell phenotypes of CD34, CD45, CD73, CD90, CD105 and HLA-DR were detected by flow cytometry. The osteoblasts, cartilage and adipocytes After 2 weeks, the cells were stained with alizarin red, toluidine blue and oil red O, respectively. The ability of adipogenesis, osteogenesis and cartilage formation was observed. According to the International Society of Stem Cell Therapy (ISCT) criteria for mesenchymal stem cells (MSCs), the isolated cells were evaluated comprehensively. P2 generation cells were cultured in DMEM 10% serum medium with different p H values (6.2, 6.5, 6.8, 7.1, 7.4) in a 37 ° C, 21% O2, 5% Cell viability (OD) was detected by cell counting kit-8 (CCK-8) after 9, 11 and 13 days. Cell viability was detected by flow cytometry at 3 days. Cultured medium for 28 days, the m RNAs of P2 generation “dry maintenance” genes Oct4, Nanog, Jag1, Notch1 and acid ion channel family protein genes ASIC1, ASIC2, ASIC3 and ASIC4 were detected by real-time fluorescence quantitative polymerase chain reaction Express the situation. Results: All the cells in P0 generation grew adherently. The immunophenotypic identification of P2 generation showed that the expression rates of CD90, CD105 and CD73 on MSCs surface were up to 96%, 95% and 94% respectively, and the expression of CD45, CD34 and HLA-DR was lower than 4%. Alizarin red staining, oil red O staining and toluidine blue staining confirmed that all the isolated cells could differentiate into three lines of bone, fat and chondrocytes. According to the ISCT criteria for MSCs, NPMSCs were isolated. The results of cell metabolic activity showed that there was no significant difference in cell OD value among P2 generation cells at 1 and 3 days after culture (P> 0.05). The OD values of cells at 5 days, 7 days, 9 days, 11 days and 13 days were statistically different (P <0.05), and the lower the value of p H, the smaller the OD value of cells. The apoptotic rate of pH 7.4 group was less than that of other groups, with statistical significance (P <0.05). The m RNAs of pH 7.4, “dry maintenance” genes Oct4, Nanog, Jag1, Notch1 and acid ion channel family protein genes ASIC1, ASIC2, ASIC3 and ASIC4 were significantly higher than those of pH values 7.1, 6.8, 6.5, Group 6.2 (P <0.05). With the decrease of p H in culture medium, the apoptosis rate of cells increased, and the expression of m RNA in cells with dry maintenance and acid channel family proteins decreased. CONCLUSION: The culture of adult NPMSCs with low p H value for 1 and 3 days had no significant effect on the proliferation of adult NPMSCs. After 5 days of culture, the proliferation and gene expression of NPMSCs were significantly inhibited and the apoptosis of NPMSCs was promoted.