不同浓度尿酸对人类髓核细胞生物学功能的影响

Effects of different concentrations of uric acid on biological functions of human nucleus pulposus cells

目的 探讨不同浓度尿酸对人类髓核细胞(HNPC)生物学功能的影响。方法 体外不同浓度尿酸条件下培养HNPC,采用CCK-8法检测不同浓度尿酸对HNPC的抑制率。分别采用不同浓度(0、100、200、400μmol/L)尿酸培养HNPC 1周或2周,TUNEL荧光评估HNPC凋亡水平,qRT-PCR及免疫组织化学染色检测HNPC细胞外基质Ⅱ型胶原和聚集蛋白聚糖的表达,Western blot法检测凋亡相关蛋白和自噬相关蛋白的表达。结果 随着尿酸浓度的增加,HNPC活力呈递减趋势。培养1周后,与尿酸0μmol/L比较,尿酸400μmol/L HNPC中凋亡细胞比例和切割型Caspase-3、Beclin1、微管相关蛋白轻链3(LC3)Ⅱ/LC3Ⅰ比值表达增高(P<0.05),而p62蛋白、Ⅱ型胶原及聚集蛋白聚糖mRNA和蛋白表达降低(P<0.05);培养2周后,与尿酸0μmol/L比较,尿酸200μmol/L、尿酸400μmol/L上述指标改变均较显著(P<0.05)。结论 高浓度尿酸可以促进HNPC凋亡以及自噬,减少HNPC细胞外基质表达,进而加重椎间盘退变。

Objective To explore the effects of different concentrations of uric acid on the biological functions of human nucleus pulposus cells(HNPC).Methods HNPC was cultured in different concentrations of uric acid in vitro,and the inhibition rate of HNPC in different concentrations of uric acid was measured by CCK-8method.HNPC was cultured with uric acid in different concentrations of uric acid 0,100,200,and 400μmol/L for 1or 2 weeks.TUNEL fluorescence was used to evaluate the apoptosis level of HNPC.qRT-PCR and immunohistochemical staining were used to detect the expressions of typeⅡcollagen and aggregation proteoglycan in the extracellular matrix of HNPC.Western blot was used to detect the expressions of apoptosis-related protein and autophagy-related protein.Results The activity of HNPC decreased with the increase of uric acid concentration.After 1 week of culture,compared with uric acid 0μmol/L group,the proportion of apoptotic cells and the expressions of Caspase-3,Beclin1,microtubule-associated protein light chain 3(LC3)Ⅱ/LC3Ⅰratio in HNPC were increased,while the expressions of p62protein,typeⅡcollagen and aggrecan mRNA and protein were decreased in uric acid 400μmol/L group(P<0.05).After 2weeks of incubation,compared with uric acid 0μmol/L group,the above indexes were changed significantly in uric acid 200μmol/L group and uric acid 400μmol/L group(P<0.05).Conclusion High concentration of uric acid can promote the apoptosis and autophagy of HNPC,reduce the expression of extracellular matrix of HNPC,and then aggravate the degeneration of intervertebral disc.