O-GlcNAc糖基化修饰诱导髓核细胞自噬作用机制的体外实验

Study on the Mechanism of Autophagy of Nucleus Pulposus Cells Induced by O-GlcNAc Glycosylation Modification

目的:观察髓核(NP)细胞O-GlcNAc糖基化修饰对其自噬的调节作用。方法:选取12周龄健康雄性SD大鼠,提取NP细胞进行分离和培养,取第二代SD大鼠NP细胞分为对照组和实验组;对照组和实验组均经过压力培养的第二代SD大鼠髓核细胞,实验组分别选用氧联乙酰葡萄糖胺转移酶(OGT组)和氧联乙酰葡萄糖胺水解酶(OGA组)进行干预;采用流式细胞技术定量分析NP细胞在OGT/OGA作用后细胞存活比例,采用实时荧光定量PCR于在0 h、12 h、 24 h、36 h及48 h时对NP细胞自噬相关基因LC3B、Beclin-1和凋亡相关基因Caspase-3、Bcl-2及Bax的表达。结果:随着培养时间延续,自噬空泡阳性细胞数目逐渐增加,36 h时达峰值,48 h后减少;OGA组与对照组比较,24 h、36 h及48 h时的阳性细胞比例高于对照组,凋亡细胞比例低于对照组,存活细胞比例高于对照组,差异有统计学意义(P<0.05);SD大鼠NP细胞内随着培养时间的延续,NP细胞中Bcl-2基因的表达减少,Caspase-3及Bax基因的表达增多,差异有统计学意义(P<0.05);随着培养时间的延续,NP细胞中LC3B与Beclin-1基因的表达增加,36 h达峰值,48 h降低,差异有统计学意义(P<0.05)。结论:OGA能够通过抑制O-GlcNAc糖基化修饰过程来促进NP细胞自噬过程的发生,进而对NP细胞凋亡过程发挥抑制作用。

Objective: To observe the regulation of autophagy by O-GlcNAc glycosylation modification in nucleus pulposus(NP) cells. Methods: Primary NP cells were isolated from12-week old healthy male Sprague-Dawley rats and cultured. The second generation of primary NP cells were divided into one control group and two experimental group and cultured under pressure. One experimental group was treated with oxydiacetyl-glucosamine transferase(OGT group) and another group was treated with oxydiacetyl-glucosamine hydrolase(OGA group). Flow cytometry was used to quantitatively analyze OGT/OGA-mediated apoptotic rates. Real-time quantitative PCR(qPCR) was used to measure mRNA levels of LC3 B, Beclin-1, Caspase-3 and Bcl and Bax. Results: The number of autophagic vacuolar positive cells gradually increased, peaked at 36 h and decreased after 48 h. The proportions of autophagic vacuolar positive cells were higher at 24 h, 36 h and 48 h in OGA group than those in the control group, while the percentage of apoptotic cells was lower in OGA group than that in the control group, and their differences were statistically significant(P<0.05). Over the time, the expression level of Bcl-2 was decreased, while the expression levels of Caspase-3 and Bax were upregulated. The differences were statistically significant(P<0.05). In addition, the expression levels of LC3 B and Beclin-1 gene were increased, peaked at 36 h and decreased at 48 h. The differences were statistically significant(P<0.05). Conclusion: OGA can promote the autophagy of NP cells by inhibiting O-GlcNAc glycosylation modification, leading to inhibition NP cellular apoptosis.