纳米氧化锌对奶牛小肠上皮细胞增殖及凋亡的影响

为研究纳米氧化锌(nano-ZnO)对奶牛小肠上皮细胞(BIECs)增殖及凋亡的影响,本实验选取初生犊牛P3代十二指肠上皮细胞为试验对象,向培养基中添加0(对照)、0.2、0.4、0.8、1.6、3.2、6.4、12.8μg/mL不同浓度的nano-ZnO培养12 h后,通过倒置显微镜、CCK-8法检测细胞的生长状态和增殖活性;微量法检测LDH漏出量;DAPI荧光染色结合Image J图像测定分析nano-ZnO对BIECs凋亡的影响;实时荧光定量PCR对细胞凋亡基因Caspase-3、Caspase-8、Caspase-9、Bax、Bcl-2 mRNA的表达进行测定。结果显示,与对照组相比,nano-ZnO低剂量各组(0.2、0.4、0.8μg/m L)细胞生长状态良好,核型正常,增殖活性增加,凋亡率无明显差异(P>0.05),LDH漏出量无差异(P>0.05);高剂量各组(1.6、3.2、6.4、12.8μg/m L)细胞皱缩变圆,密度减小,细胞核致密浓染、裂解,细胞增殖活性降低(P<0.05),凋亡率明显升高(P<0.05),LDH漏出量明显增加(P<0.05);实验组基因Caspase-3、Caspase-8、Bcl-2的相对表达量与对照组相比随着nano-ZnO浓度的增加呈现先增加后下降的趋势,Caspase-9的表达量随着nano-ZnO浓度的增加先下降后升高再下降,Bax随着Nano-ZnO浓度的增加而下降(P<0.05或P<0.01)。结论:nano-ZnO低剂量组对BIECs的增殖有促进作用;高剂量组能够抑制BIECs增殖,促进细胞凋亡。

Effect of nano-ZnO on intestinal epithelial cell proliferation and apoptotic in bovine

To study the effects of nano-ZnO on the proliferation and apoptosis of bovine intestinal epithelial cells(BIECs),primary calf P3 generation small intestinal epithelial cells were selected as test materials,and nano-ZnO were added into the medium at final concentrations of 0(control),0.2,0.4,0.8,1.6,3.2,6.4 or 12.8μg/m L and treated for 12 h.Later,the growth status and proliferation activity of the cells were observed by an inverted microscope and CCK-8 method,and the amount of LDH leakage was detected by a micro method,and the effect of nano-ZnO on the apoptosis of BIECs was analyzed by DAPI fluorescence staining combined with Image J measurement.The m RNA expressions of apoptosis genes Caspase-3、Caspase-8、Caspase-9、Bax、Bcl-2 were measured.The results showed that,compared with the control group,the cells in the low-dose nano-ZnO groups(0.2,0.4 or 0.8μg/m L)had better growth status,normal karyotype,increased proliferation activity,and there were no difference in apoptosis rate(P>0.05)and the amount of LDH leakage(P>0.05).In the high-dose groups(1.6,3.2,6.4,12.8μg/m L),the cells were shrunk and rounded,the density was decreased,the nucleus was densely stained and lysed,and the cell proliferation activity was reduced.Apoptosis rate and LDH leakage were significantly increased(P<0.05).The relative expression levels of genes Caspase-3 and Caspase-8 in the test group had a trend of increasing at first and then decreasing with the increase of nano-ZnO dose,compared with the control group.The expression of Caspase-9 was decreased at first and then increased with the increase of nano-ZnO dose.Bax was decreased with the increase of nano-ZnO dose(P<0.05)or P<0.01).In conclusion,nano-ZnO low-dose were promoted the proliferation of BIECs,but high-dose nano-ZnO were inhibited the proliferation of BIECs and promoted apoptosis.