中文摘要：

目的体外模拟退变椎间盘的酸性微环境对髓核间质干细胞及脂肪间质干细胞生物学行为的影响。方法从健康雄性大鼠腹股沟中提取的脂肪问质干细胞和尾椎间盘提取的髓核间质干细胞分别于四种不同的pH条件下进行培养：标准条件（pR7．4），模拟正常、轻度退变和重度退变的椎间盘微环境（pH分别为7．1、6．8和6．5）。采用流式AV／PI染色和CCK-8测定细胞存活和增殖，实时定量PCR和WesternBlot测定聚集蛋白聚糖、Ⅰ型胶原、Ⅱ型胶原、基质金属蛋白酶-2（matrix metalloproteinase．2，MMP-2）、IV型血小板结合蛋白基序的解聚蛋白样金属蛋白酶（a disintegrin and metalloproteinase with thrombospondin motifs-4，ADAMTS4）和组织金属蛋白酶抑制因子3（tissue inhibitor of metalloproteinase．3，TIMP-3）的基因和蛋白表达水平。结果两种干细胞的凋亡和坏死细胞比例在酸性培养条件下均出现上升，其中髓核间质干细胞的凋亡及坏死细胞比例低于脂肪间质干细胞。两种干细胞在酸性条件下培养3--5d增殖水平均出现下降。在相同的酸性培养条件下，髓核间质干细胞的增殖水平高于脂肪间质干细胞（P均〈O．05）。随着培养基pH值下降，两种干细胞的聚集蛋白聚糖、Ⅰ型胶原、Ⅱ型胶原和TIMP-3的mRNA和蛋白表达水平均下降，而MMP-2和ADAMTS4上升。同等培养条件下髓核间质干细胞中聚集蛋白聚糖和Ⅱ型胶原的mRNA和蛋白表达水平高于脂肪间质干细胞，而Ⅰ型胶原、MMP-2和ADAMTS4低于脂肪间质干细胞。结论退变椎间盘酸性微环境对脂肪间质干细胞和髓核问质干细胞的细胞存活和增殖能力有抑制作用，对髓核间质干细胞的抑制作用较弱。

英文摘要：

Objective To compare the viability, proliferation and matrix metabolism of nucleus pulpous-derived mesen- chymal stem cells （NPMSCs） compared with adipose-derived rnesenchymal stem cells （ADMSCs） under intervertebral disc （IVD）- like acidic conditions. Methods In our vivo experiment, ADMSCs and NPMSCs from Sprague Dawley rats were cultured under four different pH levels representing the standard condition （pH 7.4） and the normal, mildly degenerated and severely degenerated IVD （pH 7.1, 6.8 and 6.5, separately）. By using annexin-V-FITC/propidium iodide staining and a CCK-8 assay, we examined cell viability and cell proliferation respectively. The expression of aggreean, collagen- Ⅰ, collagen-Ⅱ, MMP-2, ADAMTS4 and TIMP-3 at mRNA and protein levels were measured by RT-PCR and Western blotting. Results Flow cytometry and CCK-8 assays revealed that the viability and proliferation of ADMSCs and NPMSCs decreased with increasing acidity from pH 7.4 to 6.5. Compared with ADMSCs, NPMSCs had significantly fewer apoptotic and necrotic cells under acidic conditions （P〈0.05）. Cell proliferation of NPMSCs was significantly greater than that of ADMSCs under IVD-like acidic conditions （P〈0.05）. RT-PCR and western blotting indicated that, in both cell types with increasing acidity, the expression of aggrecan, collagen- Ⅰ , collagen-Ⅱ and TIMP-3 at mRNA and protein levels decreased, while that of MMP-2 and ADAMTS4 increased. Compared with ADMSCs, NPMSCs expressed significantly higher levels of aggreean and collagen- Ⅱ, and lower levels of collagen-Ⅰ , MMP-2 and ADAMTS4. Conclusion An acidic environment can significantly restrain the IVD regeneration by ADMSCs or NPMSCs. NPMSCs appeared less sensitive to inhibition by acidic pH, and might be promising candidates for cell-based IVD regeneration.