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Objective To investigate the protective effects of Bcl-2 associated with athanogene-1 (BAG-1) gene on human neuroblastoma cells (SH-SY5Y) injury induced by hypoxia/reoxygenation,and its influence on heat shock protein 70 (HSP70) expression.Methods The SH-SYSY cells at logarithmic growth phase were collected.Lenti virus mediated RNA interference (RNAi) technology was used to suppress the BAG-1 expression.The cells screened out can be divided into four groups:the cell control group with no lentivirus infection,lentivirus control group (containing only fluorescein protein lentivirus infection),BAG-1 siRNA group (BAG-1 siRNA silencing group),including BAG-1 siRNA-α group and BAG-1 siRNA-β group with lentivirus containing fluorescein protein (GFP) but at different BAG-1 siRNA target sites of silencing.Western Blot was used to detect the protein expression of BAG-1 and HSP70 in target cells after infectious recombination lentivirus for 72 hours;the Cell Counting Kit-8 (CCK-8) was used to detect the activity of four different group cells after hypoxia;the flow cytometry was used to detect the cell apoptosis;the HSP70 mRNA transcription level were detected by real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-qPCR) respectively in each group.Results After lentiviral infection for 72 hours,the Western Blot results showed that in the two BAG-1 siRNA silencing groups,the interference effect of BAG-1 siRNA-β group was superior to that of BAG-1 siRNA-α group.The cell viability of each group showed an increase followed by a decrease with the prolongation of hypoxia time,and reaching the peak at 8 hours.After hypoxia for 8 hours being given,the cell viability in BAG-1 siRNA-β group was significantly lower than that of the cell control group,lentivirus control group and BAG-1 siRNA-α group (A value:0.59 ±0.09 vs.0.94±0.12,0.90± 0.11,0.91± 0.14,P ＜ 0.01);the cell apoptosis rate was obviously higher in BAG-1 siRNA-β than that in the above three groups [(34.63 ± 3.46)％ vs.(14.83 ± 3.75)％,(19.93 ± 6.49)％,(16.40± 1.18)％,all P ＜ 0.01].There were no statistically significant differences in the HSP70 protein level and mRNA transcription level between BAG-1 siRNA-3 grroup,and the cell control group,lentivirus control group and BAG-1 siRNA-α group respectively (all P ＞ 0.05).Conclusion BAG-1 gene can play a role in protection of hypoxia nerve cells,reduce the apoptosis,and its protective effect can be independent of HSP70 gene.
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Objective To investigate the protective effects of lentivirus mediated Bcl-2-associated athanogene 1L (BAG-1L) over-expression on human neuroblastoma cells (SH-SYSY) induced by hypoxia/re-oxygenation,and to study its effect on the phosphoinositide 3 kinase serine/threonine protein kinase (PI3K/AKT) pathway.Methods SH-SYSY cells were cultured in vitro,and the cells at logarithmic phase were collected,and they were divided into recombined lentiviral infection group [infected by lentivirus containing BAG-1L and green fluorescent protein (GFP) gene],vector control group (infected by lentivirus containing GFP without BAG-1L gene) and cell control group (non-infection).Western Blot was used to detect the expression of BAG-1L in target cells after infection for 48 hours.SH-SY5Y cells were subjected to hypoxia for 8 hours and re-oxygenation for 24 hours,then the cell counting kit-8(CCK-8) was used to detect the cell activity,and the apoptosis was detected by flow cytometry after allophycocyanin labeled annexin V/7-amino actinomycin D (Annexin V-APC/7-AAD) staining.Western Blot was used to detect the protein expressions of BAG-1L,heat shock protein 70 (HSP70),AKT and phosphorylated AKT (p-AKT).Results After infection for 48 hours,exogenous BAG-1L protein bands were observed in recombined lentiviral infection group,but not observed in cell control group and vector control group.After hypoxia/re-oxygenation treatment,the cell viability in recombined lentiviral infection group was significantly higher than that in cell control group and vector control group (A value:0.689 ± 0.036 vs.0.425 ± 0.013,0.400 ± 0.012),apoptosis was significantly decreased [apoptosis rate:(26.97 ± 1.82)％ vs.(36.60± 1.45)％,(35.77 ± 3.74)％],the protein levels of BAG-1L,HSP70 and p-AKT were significantly increased [BAG-1L protein (gray value):2.405 ± 0.167 vs.0.529 ± 0.141,0.601 ± 0.099;HSP70protein (gray value):0.997±0.123 vs.0.634±0.091,0.584±0.106;p-AKT protein (gray value):1.234±0.118 vs.0.661 ± 0.210,0.712 ± 0.199,all P ＜ 0.01],but the protein level of AKT was slightly increased (gray value:1.103 ± 0.269vs.0.646 ± 0.188,0.791 ± 0.326) without statistically significant differences (both P ＞ 0.05).There was no significant difference in all parameters between cell control group and vector control group (all P ＞ 0.05).Conclusion Lentivirus mediated BAG-1L gene over-expression can protect nerve cells against hypoxic injury and apoptosis,and the protective effect may be related to the activation increase of pathway on PI3K/AKT.
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Objective To investigate the influence of sperm morphology, sperm DNA fragmentation index and seminal plasma zinc on pregnancy outcome of in vitro fertilization and embryo transfer (IVF-ET).MethodsA total of 341 infertile couples underwent IVF-ET were selected from January 2016 to June 2016 in our hospital.Sperm morphology, sperm DNA fragmentation index and seminal plasma zinc level were compared according to pregnancy.ResultsIn 341 cases, 204 cases pregnancy and 137 cases of no pregnancy, with the pregnancy rate of 59.8%(204/341).Compared with the pregnancy group, the percentage of normal sperm percentage was low, the abnormal sperm index was higher in the non-pregnant group (P<0.05), the sperm DNA index was higher (P<0.05).The percentage of normal sperm, sperm DNA fragmentation index and seminal plasma zinc and pregnancy rate in sperm morphology were linear (P<0.05), And there was a negative correlation between abnormal sperm index and sperm DNA fragment index (P<0.05), and other indicators were positively correlated (P<0.05).ConclusionSperm morphology, sperm DNA fragmentation index and seminal plasma zinc levels may influence the outcome of IVF-ET.The above parameters can be used to predict pregnancy outcome of IVF-ET.
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Objective To investigate the effects of lentivirus-mediated heat shock protein 70 (HSP70) gene on calcium homeostasis and calcium channels of PC12 cells induced by ischemic and hypoxia and its mechanisms. Methods PC12 cells at logarithmic phase were collected, and they were divided into recombined lentiviral infection group [infected by lentivirus containing HSP70 and green fluorescent protein (GFP) fluorescin gene], lentivirus control group (infected by lentivirus containing GFP without HSP70 gene) and non-infection group. PC12 cells were subjected ischemia/hypoxia for 4, 8, 12, 24 hours, and the cell activity was determined by methylthiazolyl tetrazolium (MTT) assay test inorder to determine the best time for ischemia/hypoxia. The mRNA expressions of HSP70, muscle/endoplasmic reticulum Ca2+-ATP isoforms (SERCA2a, SERCA2b), ryanodine receptor 2 (RyR2), and inositol 1,4,5-triphosphate receptor 1 (IP3R1) were determined by real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and the protein expressions of HSP70, SERCA, and IP3R were determined by Western Blot at 8 hours after ischemic/hypoxia. Flow cytometry was used to determine the levels of intracellular reactive oxygen (ROS) and intracellular Ca2+ ([Ca2+]i). Results With the prolongation of time of ischemia/hypoxia, the cell viability in all groups showed an increase followed by a weakening, and peaked at 8 hours. The cell viability at 8 hours in lentiviral infection group was significantly higher than that of the non-infection group and lentivirus control group [A value (×10-2): 20.3±2.2 vs. 14.1±2.1, 15.0±1.6, both P 0.05]; the relative fluorescence intensity of ROS and [Ca2+]i in lentiviral infection group was significantly reduced (ROS: 30.54±1.23 vs. 58.03±1.97, 57.72±2.35; [Ca2+]i: 34.50±2.05 vs. 48.20±3.02, 46.80±2.75, all P < 0.01]. Conclusion Exogenous HSP70 can maintain calcium homeostasis in the endoplasmic reticulum of PC12 cells, affect the Ca2+ channel protein regulated by calcium channel IP3R and calcium pump SERCA, which may cause hypoxia/ischemia intracellular injury.