酸环境对人髓核间充质干细胞生物学活性的影响

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

中文关键词:  椎间盘退变  髓核间充质干细胞  酸环境  生物学活性

英文关键词:Intervertebral disc degeneration  Nucleus mesenchymal stem cell  Acid environment  Biological characteristics

中文摘要:

  【摘要】 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pH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pH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mRNA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pH值越低细胞的OD值越小。pH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）。pH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mRNA均明显高于pH值7.1、6.8、6.5、6.2组（P<0.05）。随着培养液pH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mRNA表达降低。结论：低pH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pH降低作用越明显。

英文摘要:

  【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.