HSPB1 Overexpression And Its Protection Against Oxidative Damage Induced By H₂O₂ In Rat Cardiomyocyte Cell Line H9c2

Object To clone human cardiac heat shock protein Bl gene, determine the effects of HSPBl on the oxidative stress in rat cardiomyocyte cell line H9c2 and to study the related mechanisms. Methods l.The RT-PCR product HSPBl (630bp) of adult human cardiac tissue was constructed into pCDNA3.1⁺, screened by EcoRI/HindIII and double sequenced. 2. After expression test of HSPB1 recombinant in 293T, it was transfected into rat cardiomyocyte cell line H9c2 and the stable transfection cell line was made by selected culture with G418. 3. Established HSPBl stable transfected rat cardiomyocyte cell line H9c2 (HSPB1 H9c2) and empty vector transfected H9c2 (CON) were cultured and treated by H₂O₂. Then the effects of HSPBl over-expression on cell morphology were observed by light microscope, LDH release and apoptosis induced by H₂O₂ were assessed. 4. Endogenous reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨ) were analyzed by JC-1 and DCFH-DA. Results l.The recombinant pCDNA3.1⁺/HSPBl was constructed successfully. Positive clones containing 630bp insert were got by screening with EcoRI and HindIII. 2. Western blot showed that pCDNA3.1⁺/HSPB1 provided a sound expression of HSPBl in both 293T and H9c2. 3. HSPBl overexpression inhibited the morphological change induced by H₂O₂ significantly. 4. LDH releasing induced by (0,500, 1000μmol/L H₂O₂ in HSPB1 H9c2 and CON were 0.396±0.017 vs. 0.390±0.009 (P>0.05), 0.505±0.030 vs. 0.657±0.022 (PO.001) , 0.547+0.027 vs. 0.661±0.011 (P<0.001) , respectively. 5. Apoptosis induced by 150μmol/L H₂O₂ in HSPBl H9c2 and CON were 4.027%±1.628% vs. 10.693%±1.122% (P<0.01) . 6. HSPBl overexpression inhibited endogenous ROS...