Effects of lentivirus-mediated heat shock protein 70 gene on calcium homeostasis in PC12 cells undergone ischemia and hypoxia

Abstract

To investigate the effects of lentivirus-mediated heat shock protein 70 (HSP70) gene on calcium homeostasis in PC12 cells undergone ischemia and hypoxia, and the mechanism involved. PC12 cells at logarithmic phase were collected, and were divided into recombination lentivirus infection group (infected by lentivirus containing HSP70 and fluorescent gene), lentivirus control group (infected by lentivirus containing fluorescin without HSP70 gene) and non-infection group. HSP70 gene and protein expressions in PC12 cells were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot. After being challenged with ischemia and hypoxia for 4 hours, the viability of cells was detected by methyl thiazolyl tetrazolium (MTT), the levels of lactic acid dehydrogenase (LDH) in cell supernatant were determined by LDH measurement test kit. The concentration of intracelluer calcium ([Ca²⁺] i ) was assayed by flow cytometer. The activities of Na⁺ -K⁺ -ATPase, Ca⁺ -Mg⁺ -ATPase and total ATPase were measured by ATPase test kits. The expressions of exogenous HSP70 gene and protein were found by RT-PCR and Western Blot in the recombination lentivirus infection group. After being challenged with ischemia and hypoxia, the viability of cells in the recombination lentivirus infection group was increased significantly as compared with the lentivirus control group and non-infection group ( A value: 0.575 ± 0.020 vs. 0.395 ± 0.014, 0.363 ± 0.045, t1 = 17.996, t2= 10.600, both P < 0.001), the levels of LDH in culture medium and the concentration of [Ca⁺] i were decreased significantly [LDH ( U/L ): 743.46 ± 23.68 vs. 935.43±34.77, 962.89 ± 26.68, t1 = 11.179, t2 = 15.044, both P < 0.001; [ Ca²⁺] i relative fluorescence: ( 31.60 ± 2.43 )% vs. (41.48 ± 3.33)%, ( 40.40 ± 3.05 )%, t1 = 5.853, t2= 5.502, both P < 0.001], and the activities of Na⁺ -K⁺ -ATPase, Ca²⁺ -Mg²⁺ -ATPase and total ATPase were increased significantly [ Na⁺ -K⁺ -ATPase (μmol × mg⁻¹× h⁻¹): 8.608 ± 0.307 vs. 6.728 ± 0.173, 6.450 ± 0.091, t1 = 9.237, P1= 0.001, t2 = 11.675, P2 < 0.001; Ca²⁺ -Mg²⁺ -ATPase (μmol×mg⁻¹ × h⁻¹): 10.523 ± 0.036 vs. 7.910 ± 0.209, 8.064 ± 0.195, t1 = 9.718, P1 = 0.001, t2= 11.535, P2 < 0.001; total ATPase (μmol×mg⁻¹× h⁻¹): 17.041 ± 0.324 vs. 14.150 ± 0.182, 13.983 ± 0.085, t1= 16.113, t2 = 17.602, both P < 0.001]. There was no statistical difference in above indexes between lentivirus control group and non-infection group. HSP70 can maintain the PC12 cells calcium homeostasis, which may be one of the important mechanisms of anti-apoptosis.