核酸适配体对岩沙海葵毒素细胞毒性的拮抗作用

Antagonistic effect of nucleic acid aptamer on cytotoxicity of palytoxin

目的 探究核酸适配体对岩沙海葵毒素(PLTX)细胞毒性的拮抗作用。方法 采用细胞毒性实验分别检测并比较PLTX对小鼠胚胎成纤维细胞株NIH-3T3、仓鼠卵巢细胞亚株CHO-K1及人永生化角质形成细胞株HaCaT的毒性作用,以及核酸适配体13-S3对PLTX细胞毒性的拮抗作用(PLTX与适配体的浓度比例均为1∶1)。采用红细胞溶血实验检测PLTX对绵羊红细胞的溶血作用,以及适配体对PLTX溶血活性的抑制作用。结果 细胞毒性实验结果显示,HaCaT细胞的IC50为(1.24±0.93)nmol/L,CHO-K1细胞的IC50为(3.95±0.78)×10^(-3)nmol/L,NIH-3T3细胞的IC50为(5.18±1.92)×10^(-3)nmol/L。与PLTX对照组比较,各浓度(9.3×10^(-4)、9.3×10^(-3)、9.3×10^(-2)、9.3×10^(-1)、9.3、93.3 nmol/L)PLTX+适配体13-S3组的HaCaT细胞存活率均升高(P均<0.05)。在12.5 nmol/L PLTX浓度下,与PLTX+适配体N7-T(节球藻毒素R的特异性适配体)组相比,PLTX+适配体13-S3组的溶血率下降更明显(P<0.05),表明适配体13-S3能够特异性地抑制PLTX对绵羊红细胞的溶血活性。结论 HaCaT细胞存活率对PLTX浓度变化(9.3×10^(-4)~93.3 nmol/L)具有较好的梯度响应,相应浓度的核酸适配体能够拮抗PLTX对HaCaT细胞的毒性作用。在12.5 nmol/L PLTX浓度下,相应浓度适配体能够有效抑制PLTX对绵羊红细胞的溶血活性,具有细胞保护作用。

Objective To explore the antagonistic effect of nucleic acid aptamer on cytotoxicity of palytoxin(PLTX).Methods The cytotoxicity of PLTX on mouse embryonic fibroblast cell line NIH-3T3,Chinese hamster ovary cell line CHO-K1 and immortalized human keratinocyte cell line HaCaT and the antagonistic effect of aptamer 13-S3 to PLTX (with the concentration ratio of 1∶1) were detected and compared by cytotoxicity experiment.The hemolytic effect of PLTX on sheep erythrocytes and the inhibitory effect of aptamer on the hemolytic activity of PLTX were detected by erythrocyte hemolysis test.Results The cytotoxicity test showed that the half inhibitory concentration (IC50) of HaCaT cells was (1.24±0.93) nmol/L,that of CHO-K1 cells was (3.95±0.78)×10^(-3)nmol/L,and that of NIH-3T3 cells was(5.18±1.92)×10^(-3)nmol/L.Compared with the PLTX control group,the survival rates of HaCaT cells were signi?cantly increased in different concentration (9.3×10^(-4),9.3×10^(-3),9.3×10^(-2),9.3×10^(-1),9.3,and 93.3 nmol/L) PLTX+aptamer13-S3 groups (all P<0.05).At the concentration of 12.5 nmol/L PLTX,compared with the PLTX+aptamer N7-T (the specific aptamer of nodularin-R) group,the hemolysis rate of PLTX+aptamer 13-S3 group decreased more significantly(P<0.05),indicating that the aptamer 13-S3 could speci?cally inhibit the hemolytic activity of PLTX on sheep erythrocytes.Conclusion The survival rate of HaCaT cells has a good gradient response to the change of PLTX concentration (9.3×10^(-4)-93.3 nmol/L),and corresponding concentration of nucleic acid aptamers can antagonize the cytotoxicity of PLTX on HaCaT cells.At a concentration of 12.5 nmol/L PLTX,the corresponding concentration of aptamer can effectively inhibit the hemolytic activity of PLTX on sheep erythrocytes and play a cytoprotective role.