柚苷对髓核间充质干细胞生物学性能的影响及其相关机制

Biological effect and mechanism of naringin on human degenerative nucleus pulposus-derived mesenchymal stem cell

中文关键词:  髓核间充质干细胞  柚苷  椎间盘退变  凋亡  PI3K/Akt信号通路

英文关键词:Nucleus pulposus-derived mesenchymal stem cells  Naringin  Disc degeneration  Apoptosis  PI3K/Akt signaling pathway

中文摘要:

  【摘要】 目的：探讨柚苷对人退变腰椎间盘来源髓核间充质干细胞（human nucleus pulposus-derived mesenchymal stem cell，hNPMSC）生物学性能的影响及其可能机制。方法：收集腰椎间盘突出症患者的退变髓核组织，分离培养hNPMSC并进行体外扩增。通过细胞形态学观察、细胞免疫表型检测及三系分化进行间充质干细胞的鉴定。取P3代hNPMSC分为对照组（正常培养基培养）、柚苷组（以含20μg/mL柚苷的培养基培养）和LY294002组（用含20μg/mL柚苷及PI3K/Akt信号通路抑制剂LY294002的培养基培养），培养6d后通过流式细胞仪检测细胞凋亡率，TUNEL荧光检测TUNEL染色阳性细胞率，Western Blot检测凋亡相关蛋白Caspase-3、Bcl-2和Bax及PI3K/Akt信号通路相关蛋白p-Akt、Akt和p53的表达，RT-PCR检测Ⅱ型胶原及蛋白多糖的mRNA表达。结果：来自人退变腰椎间盘髓核的原代细胞贴壁生长，呈不规则多边形，传代后以梭形为主；高表达干细胞相关阳性表面抗原分子CD73、CD90及CD105，低表达CD45及CD34；茜素红染色、油红O染色及甲苯胺蓝染色证实经诱导后可向骨、脂肪及软骨细胞分化，符合间充质干细胞的表型。与对照组比较，柚苷组细胞凋亡率、TUNEL染色阳性细胞率、p53、Caspase-3和Bax蛋白表达显著性下降，p-Akt及抗凋亡蛋白Bcl-2表达显著性增加（P<0.05）；LY294002干预后可以逆转这种改变（P<0.05）。与对照组比较，柚苷组hNPMSC中Ⅱ型胶原及蛋白多糖的mRNA表达显著性增加；LY294002组中Ⅱ型胶原及蛋白多糖的mRNA表达较柚苷组显著性减少（P<0.05）。结论：柚苷可通过激活PI3K/Akt信号通路减少细胞凋亡，促进hNPMSC向髓核细胞分化。

英文摘要:

  【Abstract】 Objectives: To investigate the biological effect of naringin on human degenerative nucleus pulposus-derived mesenchymal stem cell(hNPMSC), and to explore the possible mechanism. Methods: The nucleus pulposus tissue of patients with lumbar degenerated disc disease was collected, and hNPMSCs were isolated and cultured in vitro. MSC identification was performed on the isolated cells by observing cell morphology, flow cytometric immunophenotypic assay, and multilineage differentiation. The third generation hNPMSCs were divided into different groups depending on culture medium: control group in normal medium, naringin group in medium with certain concentration of naringin, LY294002 group in medium with certain concentration of naringin and LY294002(PI3K/Akt pathway inhibitor), and the hNPMSCs in each group were incubated for 6 days separately. The apoptosis was detected by flow cytometry and TUNEL staining. The protein expressions of Caspase-3, Bcl-2, Bax and PI3K/Akt signaling pathway-related proteins p-Akt, Akt and p53 were detected by Western Blot. The mRNA expressions of collagen type Ⅱ and aggrecan were detected by RT-PCR. Results: The primary cells showed adherent growth in irregular polygons and transformed into fusiform after passage. The immune phenotype showed that the stem cell-associated positive surface antigen molecules CD73, CD90 and CD105 were highly expressed, but CD45 and CD34 were low expressed. Cells showed osteogenic, chondrogenic and adipogenic differentiation after induced by alizarin red stain, Oil red O stain and toluidine blue stain respectively. Compared with the control group, the apoptosis rate, the rate of TUNEL positive cells, protein expressions of Caspase-3, Bax and p53 in the naringin group were significantly decreased, and the protein expressions of p-Akt and Bcl-2 were significantly increased(P<0.05), but the effect could be reversed by LY294002. The mRNA expressions of collagen type Ⅱ and aggrecan were significantly increased in the naringin group when compared with the control group, but the effect could be weakened by LY294002(P<0.05). Conclusions: Naringin can inhibit apoptosis by activating PI3K/Akt signaling pathway in hNPMSCs and promote differentiation into NP cells.