Résumé
Objective To investigate mRNA and protein expressions of TGF-β1 and their signifi-cance in injured spinal cord after transplantation of activated microglia. Methods Wistar rat model of spinal cord injury was made by using modified Allen's method. Then, the activated microglia was trans-planted into spinal cord injury models tu detect mRNA and protein expressions of TGF-β1 in the spinal curd. Results The mRNA and protein expressions of TGF-β1 in control group were significantly lower than that in test group (P<0.05). Conclusion Transplantation of activated microglia to the impaired spinal cord in rats can increase the mRNA and protein expressions of TGF-β1. The up-regulated expres-sion of TGF-β1 in spinal cord may play a role in promoting the recovery of spinal cord, when the activated microglia may be the main saurce of TGF-β1.
Résumé
BACKGROUND: Nuclear factor erythroid-2-related factor-2 (NF-E2-related factor-2) is an important transcription factor to regulate anti-oxidative stress reaction. Some researches indicate that NF-E2-related factor-2 can be phosphorylated by numerous members of protein kinase C family. In order to investigate generant mechanism of microwave radiation on oxidative stress injury, whether microwave radiation can influence on anti-oxidative regulating system through NF-E2-related factor-2 or not should be further studied.OBJ ECTIVE: To analyze the effect of microwave radiation on phosphorylation of NF-E2-related factor-2 and activity of protein kinase C in vascular endothelial cells.DESIGN: Observational-contrast study.SETTING : Department of Labor Hygiene, the Third Military Medical University of Chinese PLA.MATERIALS: Vascular endothelial cell strain; H332PO4; Protein-A Sepharose (Sigma Company); mono-antibody of NF-E2-related factor-2 (H-300, Santa Cruz); α-mono-antibody of protein kinase C (Santa Cruz); glass microfiber filters(Whatman Company); gel scanning system (Gel Doc 2000, Bio-Rad); liquid scintillation spectrometer (LKB-117, Sweden).METHODS: The experiment was carried out in Laboratory of Electromagnetic radiation and Biological Effect, Department of Labor Hygiene, the Third Military Medical University of Chinese PLA from March to July 2003. ① Analysis of phosphorylation of NF-E2-related factor-2: Vascular endothelial cells were cultured with DMEM medium till the period of productive growth and incubated with 32Pi for 2 hours. And then, cultured bottle was maintained in water bath at 37℃ and performed with microwave radiation in dark chamber, whose reflectivity was about zero. It was regarded as radiation group, and the average power density of radiation was 30 mW/cm2; in addition, the duration of radiation was 30 minutes.Cells did not deal with microwave radiation were regarded as control group. Phosphorylation level of NF-E2-related factor-2 was measured at 2, 4, 8 and 24 hours after radiation with immune coprecipitation-autoradiography technique and dealt with semi-quantitative analysis with gel scanning system. Cells in the control group were analyzed directly. ②Active analysis and expressional measurement of protein kinase C: Cells in the radiation group and the control group were dealt with the same cultured method, condition, radiation styles, dosage and environment as mentioned above. At 2, 4, 8 and 24 hours after radiation, cells were split to extract plasma and membrane protein. Furthermore, activity of protein kinase C was measured with r-32P-ATP labeled liquid scintillation spectrometer; gray value of protein strap was dealt with semi-quantitative analysis with gel scanning system; staining degree of plasma was observed after immunocytochemical staining of protein kinase C. In addition, cells in the control group were measured and observed directly.MAIN OUTCOME MEASURES: ① Phosphorylation level of NF-E2-related factor-2 in radiation group and control group; ②Results of active analysis and expressional measurement of protein kinase C in radiation group and control group.RESULTS: ① Phosphorylation level of NF-E2-related factor-2 in radiation group and control group: Gray value of NF-E2-related factor-2 was higher in radiation group than that in control group at 2, 4 and 8 hours after radiation.Phosphorylation level of NF-E2-related factor-2 reached the peak at four hours after radiation. In addition, results of semi-quantitative scanning analysis showed that, at 2, 4 and 8 hours after radiation, phosphorylation level of NF-E2-related factor-2 was increased 33%, 261% and 141% in radiation group as compared with that in control group,respectively (t = 2.974, 4.209, 4.047, P ＜ 0.05), and then, fallen down to normal value 24 hours later. ② Results of active analysis and expressional measurement of protein kinase C in radiation group and control group: At 2, 4 and 8 hours after microwave radiation, expression of protein kinase C in radiation group was higher than that in control group,especially at the 4 hour. In addition, at 24 hours after radiation, expression of protein kinase C recovered the normal value. Results of immunocytochemical staining showed that staining of plasma was deeper in radiation group than that in control group at 4 hours after radiation. Moreover, results of r-32P-ATP labeled liquid scintillation spectrometer also suggested that, at 2, 4 and 8 hours after radiation, activity of protein kinase C was increased 36%, 93% and 47% in radiation group as compared with that in control group, respectively (t =2.801, 3.654, 3.035, P ＜ 0.05). And then, activity of protein kinase C was decreased after 24 hours, otherwise, activity of protein kinase C reached the peak at 4 hours after radiation.CONCLUSION: Microwave radiation can strengthen the phosphorylation level of NF-E2-related factor-2 in vascular endothelial cells during a special period; meanwhile, it can also cause the increase of expression of protein kinase C.Time effect of activity of protein kinase C is coincidence with phosphorylation level of NF-E2-related factor-2.
Résumé
BACKGROUND: Nuclear factor erythroid-2-related factor-2 (NF-E2-related factor-2) is an important transcription factor to regulate anti-oxidative stress reaction. Some researches indicate that NF-E2-related factor-2 can be phosphorylated by numerous members of protein kinase C family. In order to investigate generant mechanism of microwave radiation on oxidative stress injury, whether microwave radiation can influence on anti-oxidative regulating system through NF-E2-related factor-2 or not should be further studied. OBJECTIVE: To analyze the effect of microwave radiation on phosphorylation of NF-E2-related factor-2 and activity of protein kinase C in vascular endothelial cells.DESIGN: Observational-contrast study. SETTING: Department of Labor Hygiene, the Third Military Medical University of Chinese PLA.MATERIALS: Vascular endothelial cell strain; H332PO4; Protein-A Sepharose (Sigma Company); mono-antibody of NF-E2-related factor-2 (H-300, Santa Cruz); ?-mono-antibody of protein kinase C (Santa Cruz); glass microfiber filters (Whatman Company); gel scanning system (Gel Doc 2000, Bio-Rad); liquid scintillation spectrometer (LKB-117, Sweden).METHODS: The experiment was carried out in Laboratory of Electromagnetic radiation and Biological Effect, Department of Labor Hygiene, the Third Military Medical University of Chinese PLA from March to July 2003. ① Analysis of phosphorylation of NF-E2-related factor-2: Vascular endothelial cells were cultured with DMEM medium till the period of productive growth and incubated with 32Pi for 2 hours. And then, cultured bottle was maintained in water bath at 37℃ and performed with microwave radiation in dark chamber, whose reflectivity was about zero. It was regarded as radiation group, and the average power density of radiation was 30 mW/cm2; in addition, the duration of radiation was 30 minutes. Cells did not deal with microwave radiation were regarded as control group. Phosphorylation level of NF-E2-related factor-2 was measured at 2 , 4, 8 and 24 hours after radiation with immune coprecipitation-autoradiography technique and dealt with semi-quantitative analysis with gel scanning system. Cells in the control group were analyzed directly. ② Active analysis and expressional measurement of protein kinase C: Cells in the radiation group and the control group were dealt with the same cultured method, condition, radiation styles, dosage and environment as mentioned above. At 2, 4, 8 and 24 hours after radiation, cells were split to extract plasma and membrane protein. Furthermore, activity of protein kinase C was measured with r-32P-ATP labeled liquid scintillation spectrometer; gray value of protein strap was dealt with semi-quantitative analysis with gel scanning system; staining degree of plasma was observed after immunocytochemical staining of protein kinase C. In addition, cells in the control group were measured and observed directly.MAIN OUTCOME MEASURES: ① Phosphorylation level of NF-E2-related factor-2 in radiation group and control group; ② Results of active analysis and expressional measurement of protein kinase C in radiation group and control group.RESULTS: ① Phosphorylation level of NF-E2-related factor-2 in radiation group and control group: Gray value of NF-E2-related factor-2 was higher in radiation group than that in control group at 2, 4 and 8 hours after radiation. Phosphorylation level of NF-E2-related factor-2 reached the peak at four hours after radiation. In addition, results of semi-quantitative scanning analysis showed that, at 2, 4 and 8 hours after radiation, phosphorylation level of NF-E2-related factor-2 was increased 33%, 261% and 141% in radiation group as compared with that in control group, respectively (t = 2.974, 4.209, 4.047, P