RESUMO
Objective To prepare PTD4-Cu,Zn-SOD fusion protein.Methods The recombinant plasmid of pET 1 6b-Cu,Zn-SOD and pET16b-PTD4-Cu,Zn-SOD was transformed into Escherichia coli BL21 (DE3).Isopropyl β-D-1-thiogalactopyranoside was then added at a final concentration of 0.84 mmol/L,and the cells were incubated for 4 h to induce the expression of Cu,Zn-SOD and PTD4-Cu,Zn-SOD fusion protein.Lysozyme and ultrasound were used to lyse the bacteria,the supernatant was collected for 15％ SDS-PAGE to analyze the expression of the target protein.Ni-NTA His bind resin was used to purify Cu,Zn-SOD protein and PTD4-Cu,Zn-SOD fusion protein under natural conditions.Western blot was used to identify the target protein.Results The results of Western blot showed that the purity of the target protein was about 90％,and the Cu,Zn-SOD protein with a molecular weight about 19 kDa and the PTD4-Cu,Zn-SOD fusion protein with a molecular weight about 20 kDa were found.Conclusion PTD4-Cu,Zn-SOD fusion protein is prepared successfully.
RESUMO
Aim To investigate the antidepressive-like effect of ethyl alcohol extract of Polyrhachis vicina Rog-er(EAPR),and its mechanism.Methods EAPR was prepared by ethanol extraction.Its anti-depressive effect was investigated by tail suspension test (TST) and forced swimming test (FST).Furthermore,repeated doses of reserpine was used for preparing the depres-sive rats.Results EAPR has definitely anti-depres-sive effect,as evidenced by the decreased immobility time in FST and TST at the doses of 8 and 4 g·kg -1 (P <0.05).In the repeated reserpine evoked depres-sive rats,EAPR antagonized the symptoms induced by monoamines depletion and attenuated the anhedonia, as manifested by reversed hypothermia,akinesia and sucrose consumption at the doses of 8 and 2 g·kg -1 (P <0.05,P <0.01).Neuro-chemical studies showed that AFPR significantly increased the concentration of monoamines,including 5-hydroxytryptamine (5-HT) and noradrenaline(NA)at the dose of 8 g·kg -1 (P <0.05),and had no effect on normal rats .Furthermore, EAPR increased the activity of superoxide dismutase (SOD)in serum,hippocampus and cerebral cortex at the dose of 8 g·kg -1 (P <0.05).Conclusion EA-PR possesses the definite antidep ressive properties, connected with the regulation of neurotransmitter me-tabolism and the nerve cells antioxidant effect.
RESUMO
Objective To investigate the way that nasopharyngeal carcinoma (NPC) and NPC stem cells develops resistance to cisplatin through anti-reactive oxygen species mechanism. Methods Using CCK-8 cell counting kit, we measured the half inhibitory concentration of cisplatin against NPC cellsCNE-2and NPC stem cellsCNE-2S, and compared their resistant index. We examined the differences in the reactive oxygen species (ROS) levels, total glutathi?one (GSH) levels, and total superoxide dismutase (SOD) levels between CNE-2 and CNE-2S at different concentrations of cisplatin administration (0.1,0.5 and 1.0μmol·L-1). Using q-PCR, we determined the mRNA expression level of GSS, GCLC, GCLM, SOD1 and SOD2 after 48 hours administration of cisplatin at 1 μmol · L-1. Protein expression level of SOD2 was also tested using Western Blot after 48 hours administration of cisplatin at 1μmol · L-1. Upon silencing the SOD2 in NPC cell through siRNA, Trypan blue was used to analyze cell survival after cisplatin was administrated at 1μmol · L-1. Results The inhibition concentration of cisplatin against CNE-2 was higher than that against CNE-2S (μmol · L-1:9.8 ± 1.1 vs 2.4 ± 0.6,P<0.05). ROS levels in CNE-2 and CNE-2S both rise with cisplatin administration, but ROS levels of CNE-2 before and after cisplatin treatment were both higher than those in CNE-2S (P<0.05). The total gluta?thione levels in CNE-2 and CNE-2S were both increased after 1μmol·L-1 cisplatin treatment but there is no significant dif?ference in levels of glutathione between these two cell lines. After treated with cisplatin, SOD level were increased in both CNE-2S and CNE-2, but it is higher in CNE-2S than that in CNE-2 (P<0.05). The mRNA levels of GSS, GCLC, GCLM, and SOD1 were not different significantly between in CNE-2 and in CNE-2S with or without cisplatin treatment. However, SOD2 in CNE-2S were higher than that in CNE-2 on both mRNA and protein levels (P<0.05). Silenced SOD2 disrupted the resistance of cisplatin in CNE-2S. Conclusion These data suggest that NPC stem cells (CNE-2S) enhance its drug re?sistance to cisplatin through highly expression of SOD2 which posed anti-ROS capacity.
RESUMO
Objective To observe the protective effects of le g negative pressure (NP) treatment on free radical injury from chronic ischemia of lower legs in canines. Methods By cutting off the branches of the femoral artery a nd inserting two pieces of spiral wires into the artery of canine posterior limb , we succeeded in duplicating the animal model of arterial occlusive disease in the lower extremity within two weeks. After treatment with LNP for 8 to 10 days, the blood samples of the ischemic legs were collected and the lipid peroxide (L PO) content as well as superoxide dismutase (SOD) activity were measured and ana lyzed, respectively. Results When the ischemic model was set up, PLO content inc reased while SOD activity decreased apparently, compared with those of normal [(35.594?10.753)vs. (24.800?9.011)?mol?L -1, P
