脂多糖环境下银纳米颗粒对人骨髓间充质干细胞增殖和成骨分化的影响

Effects of Silver Nanoparticles on Proliferation and Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells Under Lipopolysaccharide Environment

目的:阐明细胞炎症环境下,银纳米颗粒(AgNP)对人骨髓间充质干细胞(BMSC)增殖和成骨分化的影响及作用机制。方法:150μg/L脂多糖(LPS)建立细胞炎症模型。在细胞炎症模型条件下BMSC分为:炎症对照组、炎症AgNP组和炎症Akt抑制剂组。正常环境下BSMC分为正常对照组和正常AgNP组。各组BMSC均进行21d成骨诱导。再分别以MTT、Western blot检测各组BMSC增殖吸光度值(A值)和Caspase 3蛋白的表达;ELISA或Western blot检测各组BMSCs的骨形成蛋白-2(BMP-2)、碱性磷酸酶(ALP)含量或相对表达水平;ELISA检测各组BMSCs的总超氧化物歧化酶(Total-SOD)和丙二醛(MDA)含量;各组BMSCs均进行茜素红染色,计算光密度值(OD值)。结果:相对于正常对照组,正常AgNP组BMSCs的A值明显升高(P<0.01);Caspase 3蛋白表达降低(P<0.01);正常AgNP组BMSCs的BMP-2、ALP、Total-SOD蛋白表达或茜素红染色OD值均明显增加(P<0.01),正常AgNP组MDA表达显著降低(P<0.01)。与炎症对照组相比,炎症AgNP组BMSCs的A值明显提高(P<0.01);Caspase 3蛋白表达显著降低(P<0.01);炎症AgNP组BMP-2、ALP、Total-SOD表达或茜素红染色OD值均明显增加(P<0.01),MDA表达显著降低(P<0.01)。炎症Akt抑制剂组的BMP-2、ALP、Total-SOD、MDA表达相对于炎症AgNP组均呈相反趋势。结论:细胞炎症环境下,银纳米颗粒通过Akt信号通路提高Total-SOD等抗氧化酶的表达,促进BMSCs增殖和成骨分化。

Objective:In the inflammation environment,observe the effects of silver nanoparticles(AgNP)on proliferation,apoptosis and osteogenic differentiation of human bone marrow mesenchymal stem cells(BMSC),and its mechanism.Methods:The cellular inflammatory model was established with 150μg/L lipopolysaccharide(LPS).Under the cellular inflammatory model,to establish inflammatory control group,inflammatory AgNP group and inflammatory Akt inhibitor group.Under normal cell culture condition,to establish the normal control group and normal silver nanoparticles group.In each group,the osteogenic induction culture of BMSCs was conducted for 21d.The MTT assay and Western blot were used to detect the BMSCs proliferation absorbance value(A value)and Caspase 3 expression in each group,respectively.The ELISA assay and Western blot were used to detect the BMSCs Bone morphogenetic protein-2(BMP-2),alkaline phosphatase(ALP)expression in each group,respectively.The ELISA assay was also used to detect the BMSC stotal superoxide dismutase(Total-SOD)and malondialdehyde(MDA)content of each group.In each group,alizarin red staining was performed and the optical density was calculated,respectively.Results:Compared with the normal control group,the absorbance value of BMSCs was significantly increased in normal AgNP group(P<0.01),and expression of Caspase 3 increased decreased(P<0.01).In normal AgNP group,the expression of BMP-2,ALP,Total-SOD proteins or optical density value of alizarin red staining was significantly increased(P<0.01),and the expression of MDA increased decreased(P<0.01).Compared with the inflammatory AgNP group,the A value of BMSCs in the inflammatory Silver nanoparticles group was significantly increased(P<0.01),and expression of Caspase 3 decreased significantly(P<0.01).In the inflammatory AgNP group,the expression of BMP-2,ALP,Total-SOD proteins or optical density value of alizarin red staining was significantly increased(P<0.01),and the expression of MDA decreased significantly(P<0.01).The expression or content of BMP-2,ALP and Total-SOD proteins in the inflammatory Akt inhibitor group were opposite to those in the inflammatory AgNP group.Conclusion:The cellular inflammatory environment,the activation of Akt signaling pathway by Silver nanoparticles increased the expression of Total-SOD and other antioxidant enzymes,and reduced the expression of MDA.It further protected the BMSCs from anti-inflammatory injury and promoted the osteogenic differentiation of BMSCs.