白芨外泌体的分离及对体外人永生化角质形成细胞(Hacat)的凋亡保护研究

Bletilla striata Exosomes:Isolation and Apoptosis Protection to Human Immortalized Keratinocyte(Hacat)in vitro

探索鲜白芨(Bletilla striata)外泌体对体外人永生化角质形成细胞(Hacat)的生物活性,为其在护肤品上的开发提供数据。聚乙二醇(PEG)沉淀法提取白芨外泌体,透射电镜鉴定,BCA法测蛋白浓度;细胞计数试剂(Cell Counting Kit-8,CCK8)法检测Hacat细胞存活率,流式双染检测凋亡,蛋白印迹法检测凋亡相关通路蛋白。白芨外泌体呈茶杯状具双层膜,平均粒径69.63 nm,每克鲜白芨含外泌体浓度为5.24E+8 Particles,内含蛋白19.53μg;白芨外泌体(≥5μg/mL)可显著提高体外Hacat细胞存活率(P<0.05);10μg/mL白芨外泌体处理48h,可显著降低H_(2)O_(2)氧化造模引起的Hacat细胞早凋亡、晚凋亡和总凋亡的细胞比例(P<0.01),同时,模型组caspase-9和caspase-3表达显著上升,外泌体处理能显著降低其表达,而Bcl-2表达在组间无差异(P<0.05)。PEG沉淀法分离的白芨外泌体,可促进体外Hacat生长,抑制H_(2)O_(2)引起的细胞凋亡且与蛋白caspase-9和caspase-3相关。

The bioactivity of the exosomes from Bletilla striata(B-exo)on human immortalized keratinocytes(Hacat)in vitro was investigated to provide data for the development of skincare products.The B-exo was extracted by polyethylene glycol precipitation and identified by transmission electron microscopy.The protein concentration was measured by the BCA Protein Assay Kit.Then,the survival rate of Hacat cells,the apoptosis rate and the apoptosis-related pathway proteins were detected by the Cell Counting Kit-8(CCK8)method,flow cytometry and western blotting,respectively.The B-exo,a double-layer membrane with a teacup-like shape,had an average particle size of 69.63 nm.Each gram of the fresh Bletilla striata contained 5.24E+8 Particles of the B-exo and 19.53μg protein.The B-exo significantly increased the survival rate of Hacat cells in all treatment groups,compared to the control(P<0.05).The ratios of early,late and total apoptosis induced by H_(2)O_(2) oxidation were all significantly decreased in Hacat cells after being treated with 10μg/mL B-exo(P<0.01).In addition,the expressions of Caspase-9 and Caspase-3 protein were significantly increased in the control group,while a decrease was detected in the B-exo treatment.However,the level of BCL-2 protein expression had no difference among the groups(P<0.05).The B-exo isolated by PEG precipitation can promote Hacat cell proliferation and inhibit H_(2)O_(2)-induced apoptosis that is modulated by the changed Caspase-9 and Caspase-3 protein expression in vitro.