镁钙合金浸提液对人结肠黏膜上皮细胞中基质金属蛋白酶9和基质金属蛋白酶抑制因子3表达的影响

Effect of magnesium-calcium alloy extract on matrix metalloproteinase-9 and matrix metalloproteinase inhibitor-3 in human colonic epithelial cells

目的研究不同浓度镁钙合金浸提液对人结肠黏膜上皮细胞NCM460中基质金属蛋白酶9(MMP9)和基质金属蛋白酶抑制因子3(TIMP3)表达的影响。方法用镁钙合金制成不同浓度(体积分数10%、50%和100%)的浸提液,并将NCM460细胞制成5×10~6L^(-1)的细胞悬液,分为空白对照组及实验1、2、3组,空白对照组每孔加入含体积分数10%胎牛血清的达尔伯克改良伊格尔高糖培养基2 000μL,实验1、2、3组每孔分别加入体积分数10%、50%及100%的镁钙合金浸提液2 000μL,分别培养1、3、5 d,采用实时荧光定量聚合酶链式反应检测NCM460细胞中MMP9和TIMP3 mRNA的表达,免疫印迹法检测NCM460细胞中MMP9和TIMP3蛋白表达。结果培养1 d后,实验1、2、3组NCM460细胞中MMP9 mRNA、TIMP3 mRNA表达水平均显著低于空白对照组(P<0.05);培养3、5 d后,实验1组NCM460细胞中MMP9 mRNA表达水平仍显著低于空白对照组(P<0.05),实验2、3组NCM460细胞中MMP9mRNA表达水平均显著高于空白对照组和实验1组(P<0.05);培养5 d后,实验3组NCM460细胞中MMP9 mRNA表达水平显著高于实验2组(P<0.05)。培养3、5 d后,实验1、2、3组NCM460细胞中MMP9 mRNA表达水平均显著高于培养1 d后;培养5 d后,实验1组NCM460细胞中MMP9 mRNA表达水平与培养3 d后比较差异无统计学意义(P>0.05),实验2、3组NCM460细胞中MMP9 mRNA表达水平均显著高于培养3 d后(P<0.05)。培养1 d后,实验2、3组NCM460细胞中TIMP3 mRNA表达水平均显著高于实验1组(P<0.05)。培养3 d后,实验1、2、3组NCM460细胞中TIMP3 mRNA表达水平显著低于对照组(P<0.05),实验2组NCM460细胞中TIMP3 mRNA表达水平均显著低于实验1、3组(P<0.05)。培养5 d后,实验1、2、3组NCM460细胞中TIMP3 mRNA表达水平均显著高于对照组(P<0.05)。实验1组NCM460细胞中培养3、5 d后的TIMP3 mRNA表达水平均显著高于培养1 d后(P<0.05),培养5 d后的TIMP3 mRNA表达水平显著高于培养3 d后(P<0.05)。实验2组NCM460细胞中培养3 d后的TIMP3mRNA表达水平显著低于培养1 d后(P<0.05),培养5 d后的TIMP3 mRNA表达水平显著高于培养1、3 d后(P<0.05)。实验3组NCM460细胞中培养5 d后的TIMP3 mRNA表达水平显著高于培养1、3 d后(P<0.05)。培养5 d后,实验1组NCM460细胞中MMP9蛋白表达水平与对照组比较差异无统计学意义(P>0.05);实验2、3组NCM460细胞中MMP9蛋白表达水平均显著高于空白对照组和实验1组(P<0.05);实验3组NCM460细胞中MMP9蛋白表达水平与实验2组比较差异无统计学意义(P>0.05)。培养5 d后,实验1、2、3组NCM460细胞中TIMP3蛋白表达水平均显著高于对照组(P<0.05);实验1、2、3组NCM460细胞中TIMP3蛋白表达水平两两比较差异均无统计学意义(P>0.05)。结论高浓度的镁钙合金浸提液对NCM460细胞中的MMP9及TIMP3基因表达有一定的影响,容易导致早期炎症反应的发生,其机制可能与浸提液中的钙离子浓度有关。

Objective To investigate the effect of different concentrations of magnesium-calcium alloy extract on the expression of matrix metalloproteinase-9（ MMP9） and tissue inhibitor of metalloproteinase-3（ TIMP3） in human colonic epithelial NCM460 cells. Methods The different concentrations of extracts（ the volume fraction was 10%,50% and 100% respectively） were made with magnesium-calcium alloy. The 5 × 106 L-1 NCM460 suspension was randomly divided into control group,experimental group 1,experimental group 2 and experimental group 3. The cells in the control group were cultured by2 000 μL high glucose Dulbecco＇ s modified Eagle＇ s medium（ containing 10% volume fraction of fetal bovine serum）. The cells in the experimental group 1,2 and 3 were cultured by 2 000 μL magnesium-calcium alloy extract with volume fraction of10%,50% and 100% respectively. The expressions of MMP9 and TIMP3 mRNA in NCM460 cells was detected by real-time fluorescence quantitative polymerase chain reaction,and the expression of MMP9 and TIMP3 protein in NCM460 cells was detected by Western blot at after one,three and five days of cultivation respectively. Results The expression of MMP9 mRNA and TIMP3 mRNA in NCM460 cells of the experimental group 1,2 and 3 was significantly lower than that in the control group after one day of cultivation（ P〈0. 05）. After three and five days of cultivation,the expression of MMP9 mRNA in NCM460 cells of the experimental group 1 was significantly lower than that in the control group（ P〈0. 05）,but the expression of MMP9 mRNA in the NCM460 cells of the experimental group 2 and 3 was significantly higher than that in the control group and the experimental group 1（ P〈0. 05）. The expression of MMP9 mRNA in NCM460 cells of the experimental group 3 was significantly higher than that in the experimental group 2 after five days of cultivation（ P〈0. 05）. The expression of MMP9 mRNA in NCM460 cells of the experimental group 1,2 and 3 after three and five days of cultivation was significantly higher than that after one day of cultivation（ P〈0. 05）. There was no significant difference in the expression of MMP9 mRNA in NCM460 cells of the experimental group 1 between three and five days of cultivation（ P〈0. 05）. The expression of MMP9 mRNA in NCM460 cells of the experimental group 2 and 3 after five days of cultivation was significantly higher than that after three days of cultivation（ P〈0. 05）. The expression of TIMP3 mRNA in NCM460 cells of the experimental group 2 and 3 was significantly higher than that in the experimental group 1 after one day of cultivation（ P〈0. 05）. After three days of cultivation,the expression of TIMP3 mRNA in NCM460 cells of the experimental group 1,2 and 3 was significantly lower than that in the control group（ P〈0. 05）; the expression of TIMP3 mRNA in NCM460 cells of the experimental group 2 was significantly lower than that in the experimental group 1 and 3（ P〈0. 05）. After five days of cultivation,the expression of TIMP3 mRNA in NCM460 cells of the experimental group 1,2 and 3 was significantly higher than that in the control group（ P〈0. 05）. The expression of TIMP3 mRNA in NCM460 cells after three and five days of cultivation was significantly higher than that after one day of cultivation（ P〈0. 05）,and the expression of TIMP3 after five days of cultivation was significantly higher than that after three days of cultivation in the experimental group 1（ P〈0. 05）. The expression of TIMP3 mRNA in NCM460 cells after three days of cultivation was significantly lower than that after one day of cultivation（ P〈0. 05）,and the expression of TIMP3 after five days of cultivation was significantly higher than that after one and three days of cultivation in the experimental group 2（ P〈0. 05）.The expression of TIMP3 mRNA in NCM460 cells after five days of cultivation was significantly higher than that after one and three days of cultivation in the experimental group 3（ P〈0. 05）. After five days of cultivation,there was no significant difference in the expression of MMP9 protein in NCM460 cells between the experimental group 1 and control group（ P〈0. 05）,the expression of MMP9 protein in NCM460 cells of the experimental group 2 and 3 was significantly higher than that in the control group and the experimental group 1（ P〈0. 05）,but there was no significant difference in the expression of MMP9 protein in NCM460 cells between the experimental group 2 and 3（ P〈0. 05）. After five days of cultivation,the expression of TIMP3 protein in NCM460 cells of the experimental group 1,2 and 3 was significantly higher than that in the control group（ P〈0. 05）;but there was no significant difference in the expression of TIMP3 protein in NCM460 cells among the experimental group 1,2 and 3（ P〈0. 05）. Conclusions The high concentration of magnesium-calcium alloy extract has certain influence on the expression of MMP9 and TIMP3 gene in NCM460 cells,which may lead to the early inflammatory reaction,and the mechanism may be related to the calcium ion concentration in the extract.