Protective effect of nimodipine on PC12 cell apoptosis induced by H2O2 / 中国比较医学杂志

Obejective To explore the protective effect of nimodipine on PC12 cell apoptosis induced by hydrogen peroxide (H2O2).Methods The cells were randomized into five groups: normal group,model group (200 μmol/L H2O2),nimodipine low-,medium-and high-dose groups (1,10,100 μmol/L nimodipne+200 μmol/L H2O2).Cell viability was measured by MTT assay.Cell apoptosis was assessed by Hoechst staining.The activity of cysteinyl aspartate specific proteinase 3 (caspase 3),caspase 9 and superoxide dismutase (SOD),the level of malonic aldehyde (MDA) were measured by colorimetry.The expression of B-cell lymphoma-2 (Bcl-2),Bcl-2 associated X protein (Bax) and p53 were detected by western blot.Results Compared with the normal group,the cell apoptosis rate,the activity of caspase 3 and caspase 9,the MDA level,and the expression of Bax and p53 were significantly increased,the cell viability,SOD activity,and expression of Bcl-2 were decreased in the model group (P<0.01).Compared with the model group,the cell apoptosis rate,the activity of caspase 3 and caspase 9,the level of MDA,and the expression of Bax were decreased;and the cell viability,SOD activity,and the expression of Bcl-2 were increased in the nimodipine low-,medium-and high-dose groups (P<0.01).Conclusions Nimodipine suppresses cell apoptosis induced by H2O2 in PC12 cells,which may be related to regulation of expression of cell apoptosis-related proteins.

Establishment of ischemic precondition model and the protective effect of nitric oxide on PC12 cell line / 中华急诊医学杂志

Objective To establish the ischemic precondition ([PC) model of PC12 cell line in vitro, and to explore the effect of nitric oxide (NO) on the IPC cerebral protection. Method PC12 cells were cultured and used for producing the model of ischemie precondition by the way of oxygen-glucose deprivation. Twenty dishes of cells were randomly divided into four groups (5 dishes for each group): control group, ischemic precondition group (IPC),non-ischemic precondition group (NIPC) and L-NAME treatment group (L-NAME). In control group, the cells were in-cubated with low glucose (<1 g/L) and2% FBS medium in normal oxygen; in IPC group, the cells were administrated with oxygen-glucose deprivation (OGD) for 6 hours, and then subjected with reperfuaion before OGD 15 hours; in NIPC group, the cells were treated the same as control group for 6 hours, and then subjected with reperfusion before OGD 15 hours; in L-NAME group, the cells received L-NAME (1 mmol/L) and cocultured for 30 minutes before OGD 6 hours, and then received the same treatment as the IPC group. To test whether the model was established, metabolic rate of MIT, LDH release were measured and the apoptosis rate was detected by flow cytometry following oxygen-glucose deprivation 15 hours. The activity of nitric oxide synthases (NOS) was as-sessed by biochemical assay. One-way ANOVA and LSD multiple comparison test were used to analyze differences among different groups, and P<0.05 was considered different. Results Compared with NIPC group, the metabolic rate of MTT increased (94.9%±35.1%, P<0.05), while LDH release and the cell apoptotic rate decreased significantly in IPC group (279.1%±28.1%, P<0.03). Compared with control group(100.0%± 13.5%),the activities of NOS increased both in NIPC and IPC groups (390.0%±14.6%, P<0.01;126.3% ±10.6%, P<0.01). Moreover, the apoptosis rates in each group (control group, IPC group, NIPC group and L-NAME group) were 5.90, 8.73, 38.62 and 11.73%,respectively. Conclusions IPC reduces the death and apoptosis rate of PC12 cell after oxygen-glucose deprivation injury. NO might be involved, but it is not the only factor.

Knocking-down of Nogo-A Gene Expression in PC12 Cell Line by Plasmid-based RNAi / 华中科技大学学报（医学）（英德文版）

To study the inhibitory effect of Nogo-A shRNA on cell line PC12, the Nogo-A shRNA (short hairpin RNA, or shRNA) was designed and synthesized. The annealed shRNA template was inserted into plasmid pGenesil-1 containing enhanced green fluorescent protein (EGFP) gene by gene cloning technique to generate eukaryotic expression vector. The recombinant plasmid was transfected into PC12 cells by lipofecamine2000 and the mRNA and protein expression level of Nogo-A gene was detected by RT-PCR and Western blotting 48 h after the transfection. Gene sequencing showed that that the Nogo-A shRNA eukaryotic expression vector was successfully constructed. No significant change was found in the Nogo-A mRNA and protein expression level in empty vector-transfected group as compared with controls (P＞0.05), while the expression level in shRNA-transfected group decreased significantly (P＜0.05). It is concluded that the pGenesil-1/Nogo-AshRNA recombinant plasmid can effectively suppress the expression of Nogo-A gene in PC 12 cells.

A novel peptides, like nerve growth factor, inducing pheochromocytoma PC12 cell differentiation / 吉林大学学报(医学版)

Objective: To isolate the key domain of a novel polypeptide fragment from NGN-β that functions like intact NGF molecule.Methods: NGF-β had been treated with cyanogen bromide and trypsin skillfully. The peptide fragment with the activity inducing PC12 pheochromocytoma cells differentiation was isolated and purified by Sephadex G-50 gel filtration chromatography , DE-52 celluloseion exchange chromatography and C-18 reversed-phase column HPLC after NGF-β cleavaged by CNBr at 9th met then by trypsin at Arg or Lyscleavaged by CNBr at 9th met then by trypsin at Arg or Lys. Amino acid sequencing of this novel peptide fragment was performed by Automatic Amino Acid Analyser and Amino Acid Sequencer. Results: The functional fragment from cleavaged NGF might induce differentiation of PC12 cells. The fragment was consisting of two linear polypeptides . One of them was 16 peptide, GEFSVCDSVSVWVGDK , and other was 14 peptide, HWNSYCTTTHTFVK, linked by a disulphide bridge corresponding to residues 10～25 and 75～88, respectively, of the amino acid sequence of nerve growth factor, the result of biological activity assay in PC12 cells showed that the optimum concentration of this peptide were 0.001～0.1 μg*L-1. Conclusion: A novel peptide inducing differentiation of PC12 cell line of pheochromocytoma cells was obtained in the study. It′s isolation and purification successfully will underlie synthesis or expression of hyperactive neurotrophic small molecular substance although the relationship between the configuration and functions is not clearly.

THE STUDY OF NEUROPROTECTION OF bcl-2 GENE BY TRANSFECTING IT INTO PC12 CELL LINE / 解剖学报

Objective To investigate the biological effects of bcl-2 gene on neurons. Methods The recombinant expression plasmid pc DNA3-bcl-2 was constructed from pSFFV-bcl-2,then it was introduced into PC12 cell line by liposome method.Western blotting and immunohistochemistry in situ were applied to exam the exogenous gene expression.The two groups of cells(Group A,PC12 transfected by pcDNA3-bcl-2 and Group B,PC12 transfected by pcDNA3) were exposed to cisplatin with the concentration of 10*!?mol/L,50*!?mol/L, and 100*!?mol/L 72 hours later,the survival cells were estimated.Cell cycle indexes between these two groups were also studied by FCM. Results The recombinant expression plasmid pcDNA 3-bcl-2 was constructed successfully and PC12 cell line transfected by the plasmid could express Bcl-2 protein effectively.Compared with the control(25 79%),there was a significant decrease of cells from the S phase in PC12 with bcl-2 gene(8.81%).After exposed with 10*!?mol/L,50*!?mol/L,and 100*!?mol/L cisplatin,the surviving cells in group A were 276?13,185?11 and 108?10 respectively,which increased much more than in group B while they were 100?9,12?3 and 2?2 accordingly.Conclusions bcl-2 can protect PC12 cells against cytotoxic insults of cisplatin,and it suggested that it might act via cell cycle controlling.