Objective: To construct a PC12 cell strain with neuronal differentiation, and observe the apoptosis and pro- liferation activity effects induced these cells by Amyloid beta-Protein (Aβ-43). Methods: 1) PC12 cells in...Objective: To construct a PC12 cell strain with neuronal differentiation, and observe the apoptosis and pro- liferation activity effects induced these cells by Amyloid beta-Protein (Aβ-43). Methods: 1) PC12 cells in logarithmic growth phase were subcultured for 24 h. After the culture fluid was changed, the cells were treated with Rat-β-NGF and cultured for 9 days. 2) Neuronal differentiation of PC12 cells in logarithmic growth phase were divided into four groups: control group (0), experimental group (1), experimental group (2) and experimental group (3). The concentrations of Aβ in the four groups were 0 μmol/L, 1.25 μmol/L, 2.5 μmol/L and 5 μmol/L, respectively. The cells were harvested at 24, 48 and 72 h later and stained with AnnexinV-FITC/PI after centrifugation and washing. Then flow cytometry was conducted to examine the apoptosis percentage. 3) NGF-induced PC12 cells were selected and Aβ with different concentrations was added. The final concentrations of Aβ were 0 μmol/L, 1.25 μmol/L, 2.5 μmol/L and 5 μmol/L, respectively. After the cells were incubated in an atmosphere of 5% CO2 at 37 °C in an incubator for 72 h, the OD values were examined. Results: 1) Neuronal differentiated PC12 cell lines were successfully established. 2) Flow cytometric examination indicated that Aβ (1.25, 2.5, and 5.0 μmol/L) could effectively induce apoptosis of neuronal-differented cells at the 24 h, 48 h and 72 h time points. 3) Aβ (0?5.00 μmol/L) had no obvious effect on proliferation or restraining of the neuronal differentiation of the PC12 cells after a 72 h interacting process. Conclusion: This investigation revealed successful neuronal differentiation of the PC12 cell strain. The induction of apoptosis of the neurocytes by various concentrations of Aβ was observed and the in- fluence of Aβ on induced proliferation of PC12 cells by Rat-β-NGF was revealed. This study may provide basis for future research on the molecular cure of AD and interdiction of AD evolution.展开更多
BACKGROUND： The findings about the alterations in cerebrospinal fluid beta-amyloid protein （Aβ ） and apolipoprotein E （APOE） after subarachnoid hemorrhage indicate that they have significant correlation with pro...BACKGROUND： The findings about the alterations in cerebrospinal fluid beta-amyloid protein （Aβ ） and apolipoprotein E （APOE） after subarachnoid hemorrhage indicate that they have significant correlation with prognosis of patients. OBJECTⅣE： To observe the alterations in cerebrospinal fluid Aβ and ApoE after subarachnoid hemorrhage （SAH）. DESIGN： Contrast observation. SETTING： Department of Neurosurgery, the First Hospital of Lanzhou University. PARTICIPANTS： A total of 25 SAH patients including 16 males and 9 females aged from 13 to 72 years were selected form Department of Neurosurgery, the First Affiliated Hospital of Lanzhou University from October 2003 to February 2004. The Hunt-Hess grade ranged from Ⅰ to Ⅳ, and patients admitted hospital in 24 hours after invasion, affirmed by the brain CT scan and lumbar vertebra puncture, no other severe complications and important organs＇ functional defect and severe infection, no hematological system disease. METHODS- All admitted patients were collected CSF by lumbar vertebra puncture in 24 hours. The cerebrospinal fluid （CSF） of control group came from the admitted 15 patients of our hospital that have no nervous system disease. Aβ content was detected by enzyme linked immunosorbent assay （ELISA）, the kit was provided by the Central Laboratory of the First Hospital of Lanzhou University; ApoE concentration was detected by monoclone enzyme linked immunosorbent assay （ELISA）, the kit was provided by the Immunotechnique Research Institute of the Fourth Military Medical University. S100B concentration was detected by enzyme linked immunosorbent assay double antibody sandwich method, the kit was provided by the Physiological Research Room of the Fourth Military Medical University. The data were indicated on Mean±SD and were analyzed by SPSS 10.0 statistical package. All data were handled through test of significance variance analysis, and groups were compared through independent sampler t test. The concentration was handled through Pearson correlation analysis between Aβ and ApoE. The relationship between Aβ, ApoE concentration with pathogenetic condition and prognosis of the patients was handled through Spearman ranking correlation analysis. MAIN OUTCOME MEASURES：① The concentration of ApoE, Aβ and S100B after SAH in contrast to the control group in CSF by different Hunt-Hess and Glasgow Outcome Scale （GOS） grades; ② The level of correlation between ApoE and Aβ ; ③Correlation between ApoE and Aβ in pathogenetic condition and prognosis of the patients. RESULTS： All 25 SAH patients and 15 controls were involved in the final analysis. ① The concentration of ApoE, Aβ and S100B in CSF： The concentration of ApoE decreased after SAH in contrast to the control group [（0.46±0.007）, （0.85±0.11） μg/L, P 〈 0.01], the concentration of ApoE decreased after SAH in contrast to the control group [（5.36± 1.19）, （8.41± 1.60） μg/L, P 〈 0.01], and the concentration of S100B increased after SAH in contrast to the control group [（18.60±7.31）, （6.56±1.02） pg/L, P 〈 0.01]. ② The concentration of ApoE, Aβ and S100B in CSF after SAH on different Hunt-Hess and GOS grades： The concentration of Aβ in Hunt-Hess Ⅰ -Ⅲ grade was higher than Hunt-Hess Ⅳ, Ⅴ grade [（6.63 ± 1.25）, （3.35± 1.02） μg/L, P 〈 0.01], and the concentration of ApoE in Hunt-Hess Ⅰ- Ⅲ grade was higher than Hunt-Hess Ⅳ, Ⅴ grade [（0.56±0.07）, （0.38±0.04） μg/L, P 〈 0.05], the concentration of S100B in Hunt-Hess Ⅰ - Ⅲ grade was lower than Hunt-Hess Ⅳ - Ⅴ grade [（16.32±5.58）, （22.85±8.10） pg/L, P 〈 0.01]; the concentration of Aβ in GOS Ⅰ - Ⅲ grade was lower than GOS Ⅳ, Ⅴ grade [（3.76± 1.04）, （5.89±1.20） μg/L, P 〈 0.01], and the concentration of ApoE in GOS Ⅰ - Ⅲ grade was lower than GOS Ⅳ, Ⅴ grade [（0.32±0.02）, （0.58±0.07） μg/L, P 〈 0.011, and the concentration of S100B in GOS Ⅰ - Ⅲ grade was higher than GOS Ⅳ, Ⅴ grade [（25.36±9.70）, （14.33±6.69） pg/L, P 〈 0.01].③ The results of Pearson correlation analysis and Spearman ranking correlation analysis： There was significantly positive correlation between CSF Aβ concentration and clinical outcome （r=0.65, P 〈 0.01）, and the decrease in CSF Aβ concentration correlated significant with that of ApoE （r =0.85, P 〈 0.01）. CONCLUSION： There is a significant decrease in both Aβ and ApoE in the CSF after SAH, and there is significant correlation between CSF Aβ and ApoE concentration with clinical outcome, the interactions between these proteins may have important effects on SAH, ApoE and Aβ as surrogate markers for the outcome of patients with SAH.展开更多
文摘Objective: To construct a PC12 cell strain with neuronal differentiation, and observe the apoptosis and pro- liferation activity effects induced these cells by Amyloid beta-Protein (Aβ-43). Methods: 1) PC12 cells in logarithmic growth phase were subcultured for 24 h. After the culture fluid was changed, the cells were treated with Rat-β-NGF and cultured for 9 days. 2) Neuronal differentiation of PC12 cells in logarithmic growth phase were divided into four groups: control group (0), experimental group (1), experimental group (2) and experimental group (3). The concentrations of Aβ in the four groups were 0 μmol/L, 1.25 μmol/L, 2.5 μmol/L and 5 μmol/L, respectively. The cells were harvested at 24, 48 and 72 h later and stained with AnnexinV-FITC/PI after centrifugation and washing. Then flow cytometry was conducted to examine the apoptosis percentage. 3) NGF-induced PC12 cells were selected and Aβ with different concentrations was added. The final concentrations of Aβ were 0 μmol/L, 1.25 μmol/L, 2.5 μmol/L and 5 μmol/L, respectively. After the cells were incubated in an atmosphere of 5% CO2 at 37 °C in an incubator for 72 h, the OD values were examined. Results: 1) Neuronal differentiated PC12 cell lines were successfully established. 2) Flow cytometric examination indicated that Aβ (1.25, 2.5, and 5.0 μmol/L) could effectively induce apoptosis of neuronal-differented cells at the 24 h, 48 h and 72 h time points. 3) Aβ (0?5.00 μmol/L) had no obvious effect on proliferation or restraining of the neuronal differentiation of the PC12 cells after a 72 h interacting process. Conclusion: This investigation revealed successful neuronal differentiation of the PC12 cell strain. The induction of apoptosis of the neurocytes by various concentrations of Aβ was observed and the in- fluence of Aβ on induced proliferation of PC12 cells by Rat-β-NGF was revealed. This study may provide basis for future research on the molecular cure of AD and interdiction of AD evolution.
文摘BACKGROUND： The findings about the alterations in cerebrospinal fluid beta-amyloid protein （Aβ ） and apolipoprotein E （APOE） after subarachnoid hemorrhage indicate that they have significant correlation with prognosis of patients. OBJECTⅣE： To observe the alterations in cerebrospinal fluid Aβ and ApoE after subarachnoid hemorrhage （SAH）. DESIGN： Contrast observation. SETTING： Department of Neurosurgery, the First Hospital of Lanzhou University. PARTICIPANTS： A total of 25 SAH patients including 16 males and 9 females aged from 13 to 72 years were selected form Department of Neurosurgery, the First Affiliated Hospital of Lanzhou University from October 2003 to February 2004. The Hunt-Hess grade ranged from Ⅰ to Ⅳ, and patients admitted hospital in 24 hours after invasion, affirmed by the brain CT scan and lumbar vertebra puncture, no other severe complications and important organs＇ functional defect and severe infection, no hematological system disease. METHODS- All admitted patients were collected CSF by lumbar vertebra puncture in 24 hours. The cerebrospinal fluid （CSF） of control group came from the admitted 15 patients of our hospital that have no nervous system disease. Aβ content was detected by enzyme linked immunosorbent assay （ELISA）, the kit was provided by the Central Laboratory of the First Hospital of Lanzhou University; ApoE concentration was detected by monoclone enzyme linked immunosorbent assay （ELISA）, the kit was provided by the Immunotechnique Research Institute of the Fourth Military Medical University. S100B concentration was detected by enzyme linked immunosorbent assay double antibody sandwich method, the kit was provided by the Physiological Research Room of the Fourth Military Medical University. The data were indicated on Mean±SD and were analyzed by SPSS 10.0 statistical package. All data were handled through test of significance variance analysis, and groups were compared through independent sampler t test. The concentration was handled through Pearson correlation analysis between Aβ and ApoE. The relationship between Aβ, ApoE concentration with pathogenetic condition and prognosis of the patients was handled through Spearman ranking correlation analysis. MAIN OUTCOME MEASURES：① The concentration of ApoE, Aβ and S100B after SAH in contrast to the control group in CSF by different Hunt-Hess and Glasgow Outcome Scale （GOS） grades; ② The level of correlation between ApoE and Aβ ; ③Correlation between ApoE and Aβ in pathogenetic condition and prognosis of the patients. RESULTS： All 25 SAH patients and 15 controls were involved in the final analysis. ① The concentration of ApoE, Aβ and S100B in CSF： The concentration of ApoE decreased after SAH in contrast to the control group [（0.46±0.007）, （0.85±0.11） μg/L, P 〈 0.01], the concentration of ApoE decreased after SAH in contrast to the control group [（5.36± 1.19）, （8.41± 1.60） μg/L, P 〈 0.01], and the concentration of S100B increased after SAH in contrast to the control group [（18.60±7.31）, （6.56±1.02） pg/L, P 〈 0.01]. ② The concentration of ApoE, Aβ and S100B in CSF after SAH on different Hunt-Hess and GOS grades： The concentration of Aβ in Hunt-Hess Ⅰ -Ⅲ grade was higher than Hunt-Hess Ⅳ, Ⅴ grade [（6.63 ± 1.25）, （3.35± 1.02） μg/L, P 〈 0.01], and the concentration of ApoE in Hunt-Hess Ⅰ- Ⅲ grade was higher than Hunt-Hess Ⅳ, Ⅴ grade [（0.56±0.07）, （0.38±0.04） μg/L, P 〈 0.05], the concentration of S100B in Hunt-Hess Ⅰ - Ⅲ grade was lower than Hunt-Hess Ⅳ - Ⅴ grade [（16.32±5.58）, （22.85±8.10） pg/L, P 〈 0.01]; the concentration of Aβ in GOS Ⅰ - Ⅲ grade was lower than GOS Ⅳ, Ⅴ grade [（3.76± 1.04）, （5.89±1.20） μg/L, P 〈 0.01], and the concentration of ApoE in GOS Ⅰ - Ⅲ grade was lower than GOS Ⅳ, Ⅴ grade [（0.32±0.02）, （0.58±0.07） μg/L, P 〈 0.011, and the concentration of S100B in GOS Ⅰ - Ⅲ grade was higher than GOS Ⅳ, Ⅴ grade [（25.36±9.70）, （14.33±6.69） pg/L, P 〈 0.01].③ The results of Pearson correlation analysis and Spearman ranking correlation analysis： There was significantly positive correlation between CSF Aβ concentration and clinical outcome （r=0.65, P 〈 0.01）, and the decrease in CSF Aβ concentration correlated significant with that of ApoE （r =0.85, P 〈 0.01）. CONCLUSION： There is a significant decrease in both Aβ and ApoE in the CSF after SAH, and there is significant correlation between CSF Aβ and ApoE concentration with clinical outcome, the interactions between these proteins may have important effects on SAH, ApoE and Aβ as surrogate markers for the outcome of patients with SAH.
