顺铂对人骨肉瘤SaOS-2细胞核苷酸结合寡聚域1和2表达的影响

Effect of cisplatin on NOD1 and NOD2 expression in SaOS-2 cell line

目的通过观察顺铂对人骨肉瘤SaOS-2细胞核苷酸结合寡聚域1(NOD1)和NOD2表达的影响,探讨顺铂治疗骨肉瘤的作用机制。方法SaOS-2细胞加入顺铂,浓度依次为0μmol/L、5μmol/L、10μmol/L、20μmol/L,命名为S0组、S5组、S10组、S20组,培养24h、48h、72h,分别采用CCK8法、实时荧光定量聚合酶链式反应(PCR)法和免疫荧光法测定细胞生长活率、NOD1和NOD2的表达。结果在细胞培养48h、72h,S5组细胞存活率显著低于S0组(65.53%比100.00%;46.43%比100.00%,χ^2=8.64、73.97,均P<0.01)。在细胞培养24h、48h、72h,S10组、S20组细胞存活率显著低于S0组(80.60%比100.00%、42.94%比100.00%、27.90%比100.00%;62.54%比100.00%、33.09%比100.00%、22.95%比100.00%,χ^2=20.99、79.72、112.50;45.40、67.56、125.20,均P<0.01),且S20组骨肉瘤细胞存活率显著低于S5组(62.54%比93.78%、33.09%比65.53%、22.95%比46.43%,χ^2=28.47、21.78、11.71,均P<0.01)。S5组NOD1和NOD2mRNA的表达量在骨肉瘤细胞培养48h、72h显著高于培养24h且高于S0组[(3.64±0.44)比(4.47±1.22)比(1.79±0.44)比(1.00±0.00);(6.88±2.79)比(6.86±2.40)比(2.29±0.70)比(1.00±0.00),F=29.12、24.11,均P<0.01]。顺铂作用细胞48h和72h,NOD1蛋白表达较S0组有升高趋势。NOD1和NOD2mRNA的表达量呈现显著直线正相关(n=36,r=0.92,P<0.01)。结论顺铂对骨肉瘤细胞的功能有提升作用,呈现时间、浓度依赖性,顺铂可能是治疗骨肉瘤有效的药物,其机制可能是通过NOD1和NOD2途径。

Objective To investigate the effects of cisplatin on the expression of nucleotide-binding oligomerization domain-like receptor(NOD) 1 and 2 in human osteosarcoma SaOS-2 cell line, and to explore the mechanism of cisplatin in the treatment of human osteosarcoma. Methods CCK-8 assay, real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR) and immumofluorescence methods were used to determine the growth survival rate and expression levels of NOD1 and NOD2 in osteosarcoma SaOS-2 cell line treated with cisplatin (0, 5, 10, 20 μmol/L, named group S0, group S5, group S10, group S20, respectively) for 24, 48, 72 hours. Results After treatment with cisplatin for 48 h or 72 h, the growth survival rates of SaOS-2 cells were significantly decreased in group S5 than those in group S0(65.53% vs.100.00%;46.43% vs.100.00%, χ^2=8.64, 73.97, all P<0.01). Moreover, after treatment with cisplatin for 24 h, 48 h or 72 h, the growth survival rates of SaOS-2 cells were significantly decreased in group S10 or group S20 than those in group S0(80.60% vs.100.00%, 42.94 vs.100.00%, 27.90% vs.100.00%;62.54% vs.100.00%, 33.09% vs.100.00%, 22.95% vs.100.00%, χ^2=20.99, 79.72, 112.50;45.40, 67.56, 125.20, all P<0.01), and the growth survival rates were significantly lower in group S20 than those in group S5(62.54% vs.93.78%, 33.09% vs.65.53%, 22.95% vs.46.43%, χ^2=28.47, 21.78, 11.71, all P<0.01). The expression levels of NOD1 mRNA and NOD2 mRNA in group S5 were significantly increased at 48 h or 72 h than those at 24 h, and were higher than group S0 when treated with 5μmol/L cisplatin[(3.64±0.44) vs.(4.47±1.22) vs.(1.79±0.44) vs (1.00±0.00);(6.88±2.79) vs.(6.86±2.40) vs (2.29±0.70) vs.(1.00±0.00), F=29.12, 24.11, all P<0.01]. And the expression levels of NOD1 protein had an increased tendency after 48 h or 72 h treatment with 5μmol/L cisplatin.Furthermore, the expression level of NOD1 mRNA was positively correlated with NOD2 mRNA(n=36, r=0.92, P<0.01). Conclusion Cisplatin can elevate the function of osteosarcoma cell in a dose- and time-dependent manners, cisplatin may act as a efficient drug to cure osteosarcoma desease, which may be related to NOD1 and NOD2 signal pathway.