目的观察蛋白质转导4型-铜锌超氧物歧化酶融合蛋白(protein transduetion domain4-Cu,Zn superoxidedismutase fusion protein, PTD4-Cu,Zn-SOD)能否穿膜进入人星形胶质细胞、能否减轻人星形胶质细胞的缺氧损伤。方法①Westem blot:从干预时间(15、30、60min)和干预浓度(0.5、1.0、2.0μmol/L)两个方面观察融合蛋白能否穿膜。②实验分组:制备细胞缺氧损伤模型,按完全随机法分为对照组、超氧化物歧化酶(superoxide dismutase,SOD)组(SOD组)和PTD442u,Zn-SOD组(PTD4-SOD组),对照组加DMEM(dulbecco’s modified eagle’s medium,DMEM)培养基(不含血清),其余两组分别加入含有Cu,Zn-SOD和PTD4-Cu,Zn-SOD终浓度为2μmol/L的DMEM培养基,三组干预时间均为1h,用AnnexinV/PI凋亡试剂盒流式测凋亡,观察融合蛋白能否减轻细胞缺氧损伤。结果①对照组各组未发现明显的蛋白条带,SOD组各个干预时间点(15、30、60min)与对照组比较,差异无统计学意义(P〉0.05);PTD4-SOD组在15、30min两个时间点与对照组比较,差异无统计学意义(P〉0.05);在干预60min时与对照组比较,差异有统计学意义(P〈0.05)。②对照组各组未发现明显的蛋白条带,SOD组的各个剂量(0.5、1.0、2.0μmol/L)与对照组比较,差异无统计学意义(P〉0.05);PTD4-SOD组在0.5、1.0μmol/L两个浓度与对照组比较,差异无统计学意义(P〉0.05);在浓度为2μmol/L时与对照组比较,差异有统计学意义(P〈0.05)。③对照组的凋亡率为(27.03±0.50)%,SOD组凋亡率为(23.29±0.58)%,和对照组比较,差异有统计学意义(P〈0.05);而PTD4-SOD组凋亡率为(12.06±0.19)%,与对照组比较,差异有统计学意义(P〈0.05);PTD4-SOD组和SOD组比较,差异有统计学意义(P〈0.01)。结论①PTD4-Cu,Zn-SOD能进入体?
Objective To test whether protein transduction domain4-Cu, Zn superoxide dismutase fusion protein (PTD4-Cu, Zn-SOD) can penetrate into human astrocytes and whether it can mitigate hypoxic damages in human astrocytes. Methods ① Western blot was employed to analyze penetration of superoxide dismutase (SOD) and PTD4-Cu, Zn-SOD into human astrocytes. ② The human astrocyte cultures were subjected to hypoxia, then were respectively treated with dulbecco's modified eagle's medium (DMEM) (Control), SOD (2 μ mol/L) and PTD4-Cu,Zn-SOD(PTD4-SOD, 2μ mol/L). Flow cytometry was performed to analyze apoptosis in human astroeytes labeled with Annexin V/PI apoptosis Kits. Results ① PTD4-SOD, but not SOD, penetrated into human astrocyte euhures after 60 rain incubation. ② Although 0.5-2.0 μmol/L SOD did not penetrate into human astrocytes, 2.0 μmol/L (not 0.5 mol/L and 1.0 μmol/L) PTD4-SOD did. ③ Hypoxia-induced apoptosis in (27.03 ±0.50)% of astrocytes. SOD and PTD4-SOD significantly reduced apoptotic cells to (23.29±0.58)% and (12.06±0.19)% of total cells, respectively. Conclusions PTD4-Cu,Zn- SOD can penetrate the membrane of human astrocytes and reduce hypoxia-induced apoptosis,depending on the incubation time and dosage.