免疫荧光法检测融合蛋白PTD4-Cu,Zn-SOD在人星形胶质细胞中穿膜能力及保护作用  

Penetration ability and biological activity of fusion protein PTD4-Cu,Zn-SOD in human astrocytes in vitro by immunofluorescence test

作　　者：孟丽华[1] 薛荣亮[1] 雷晓鸣[1] 镇路明 魏兰璎 MENG Lihua;XUE Rongliang;LEI Xiaoming;ZHEN Luming;WEI Lanying(Department of Anesthesiology,The Second Affiliated Hospital of Xi’an Jiaotong University,Xi’an 710004,China)

机构地区：[1]西安交通大学第二附属医院麻醉科,陕西西安710004

出　　处：《西安交通大学学报（医学版）》2023年第2期309-314,共6页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：国家自然科学基金资助项目(No.81071070)。

摘　　要：目的观察蛋白质转导4型-铜,锌超氧物歧化酶融合蛋白(protein transduction domain4-cuprum,zinc superoxide dismutase fusion protein,PTD4-Cu,Zn-SOD)能否穿膜进入体外培养的人星形胶质细胞,能否在穿膜后仍保持其生物学活性,能否减轻人星形胶质细胞的缺氧损伤。方法免疫组化及荧光法观察融合蛋白穿膜后在人星形胶质细胞内的荧光分布情况,评估融合蛋白的穿膜能力;制备人星形胶质细胞缺氧损伤模型后,实验分为空白对照组、Cu,Zn-SOD组、PTD4-Cu,Zn-SOD组。空白组加DMEM培养基(不含血清)作为对照,其余两组分别加入含有Cu,Zn-SOD、PTD4-Cu,Zn-SOD终浓度为2μmol/L的DMEM培养基,干预时间为1 h,用SOD活性及MDA含量测试盒,观察融合蛋白干预后的缺氧损伤星形胶质细胞中二者的变化。结果FITC荧光标记的融合蛋白PTD4-Cu,Zn-SOD可以进入体外培养的人星形胶质细胞中,且在细胞核中有聚集分布;经3组干预后,和对照组相比,PTD4-Cu,Zn-SOD组及Cu,Zn-SOD组均能提高星形胶质细胞内的SOD活性,融合蛋白PTD4-Cu,Zn-SOD组提高星形胶质细胞内SOD活性更为明显。和Cu,Zn-SOD组、对照组相比,PTD4-Cu,Zn-SOD组能降低星形胶质细胞内的MDA含量。结论PTD4-Cu,Zn-SOD能穿膜进入体外培养的人星形胶质细胞;融合蛋白PTD4-Cu,Zn-SOD能增加人星形胶质细胞缺氧损伤后的SOD活性,降低MDA含量。Objective To observe the penetration and biological activity of PTD4-Cu,Zn-SOD into human astrocytes and whether it can mitigate hypoxia damages.Methods(1)Immunohistochemistry and fluorescence test:We labeled the Cu,Zn-SOD by a monoclonal antibody,combined it with the fluorescent secondary antibody labeled with fluorescein isothiocyanate(FITC)to observe the effect of transduced PTD4-Cu,Zn-SOD on the viability of human astrocytes.(2)The experimental group:After hypoxic damage model,the cells were divided into three groups:blank control,group Cu,Zn-SOD,and group PTD4-Cu,Zn-SOD.Group blank was added with DMEM medium(excluding serum)as control;DMEM medium was added to the other two for one hour(excluding serum)with its fusion proteins(Cu,Zn-SOD and PTD4-Cu,Zn-SOD)with the final concentration of 2μmo L/L.After the intervention,we used SOD and MDA test kits to observe PTD4-Cu,Zn-SOD and Cu,Zn-SOD in astrocytes after fusion protein intervention.Results The PTD4-Cu,Zn-SOD fusion protein could have aggregation distribution in the nucleus by FITC fluorescently labeled.After the intervention,it could increase the SOD activity in astrocytes in group PTD4-Cu,Zn-SOD and group Cu,Zn-SOD compared with control group,but the SOD activity was more obvious in the fusion proteins PTD4-Cu,Zn-SOD group.And the dose of MDA was reduced in group PTD4-Cu,Zn-SOD compared with group Cu,Zn-SOD and control group.Conclusion PTD4-Cu,Zn-SOD fusion protein can transcellular membrane of human astrocytes.The fusion protein PTD4-Cu,Zn-SOD can increase the SOD activity and reduce the content of MDA by human astrocytes from hypoxia injury.

关 键 词：蛋白质转导超氧物歧化酶融合蛋白 穿膜能力 免疫荧光 

分 类 号：R319[医药卫生—基础医学]