RESUMEN
BACKGROUND: It is indicated in researches of recent years that both astragalus and angelica act on anti-free radical and protect renal injury due to ischemia / reperfusion.OBJECTIVE: To observe the protection and its mechanism of astragalus and angelica injections on adenosine triphosphate-ase (ATPase) in renal injury due to ischemia/reperfusion.DESIGN: The observing controlled experiment based on experimental animals .SETTING: Physiological teaching & research room and teaching & research room of renal functional protection in a medical college. MATERIALS: The experiment was performed in Physiological Experimental Room of Luzhou Medical College from January 2001 to March 2001. Totally 33 Japanese big-ear white healthy adult rabbits of either sex were employed,provided by Experimental Animal Center of Luzhou Medical College, in the mass of(1.63 + 0. 22) kg. According to random number table, they were divided in sham-operation control(8 rabbits), simple ischemia/reperfusion group (8 rabbits), astragalus injection + ischemia/reperfusion group (astragalus group) (8 rabbits) and angelica injection + ischemia/reperfusion group(angelica group) (9 rabbits).METHODS: One day before operation, on the day of operation and 1 day after operation, successively, intravenous medical injections (astragalus 1.25 g/kg,angelica 12.5 g/kg) were administrated in astragalus and angelica groups everyday respectively, and injection with physiological saline 5 mL/kg was applied in the control and simple ischemia/reperfusion group. In 48 hours reperfusion after 1 hour ischemia in kidney, blood sample was collected from inferior vena cava. The upper tissue of the right kidney was collected and fixed by placed in 30 mL/L glutaraldehyde and the lower tissue was prepared into homogenate. Ultrastructure of renal tissue was examined with electron microscope; serum creatinine level and ATPase activity in renal tissue were assayed.MAIN OUTCOME MEASURES: Ultrastructure of renal tissue, serum creatinine level and ATPase activity in renal tissue.RESULTS: In simple ischemia/reperfusion group, renal tissue was degenerated significantly, and the disorders in astragalus and angelica groups were reduced markedly compared with simple ischemia/reperfusion group. Serum creatinine level in simple ischemia/reperfusion group was higher remarkably than the sham-operation control ( P ＜ 0. 05 ), and that in astragalus and angelica groups was reduced than simple ischemia/reperfusion group (P ＜ 0. 05) . In simple ischemia/reperfusion group, the levels of Mg2+-ATPaes, Na+-K+-ATPase and Ca2+-ATPase were(0. 155 ±0. 020),(0.179±0.018), (0.150±0.022) nkat/g respectively, which was markedly reduced compared with sham-operation control [ (0. 174 + 0. 012),(0. 198 + 0. 012), (0. 181 + 0. 017) nkat/g], ( t = 2. 344, 2. 438, 3. 014,P ＜ 0.05 ). In astragalus and angelica groups, respectively, the activities of Mg2+-ATPaes, Na+-K+-ATPase and Ca2+-ATPase were(0. 172 ± 0. 023),(0. 196 ±0. 077), (0. 175 ±0. 016) and (0. 177 ±0. 015), (0. 200 ±0.011 )and (0. 181 ± 0. 025) nkat/g successively. Except that Mg2+-ATPaes activity in astragalus group was not different significantly from that in simple ischemia/reperfusion group, all the rest were higher than simple ischemia/reperfusion group(t =2. 372 -2. 786, P ＜0.05).CONCLUSION: Both astragalus and angelica inhibit the decrease of ATPase and improve the disturbed local blood-flow adjustment in kidney, which has provided experimental basis of astragalus and angelica on reducing renal injury induced by ischemia/reperfusion through protecting ATPase.
RESUMEN
AIM: To clone mouse pdx-1 gene and construct its eukaryotic expression vector for expression of pdx-1 in mouse embryonic stem cells.METHODS: Mouse pdx-1 cDNA fragment was amplified with polymerase chain reaction (PCR) from mouse pancreatic cDNA. The purified fragment was recombinated with a eukaryotic expression vector carrying enhanced green fluorescent protein, pEGFP-N1. The pdx-1 cDNA fragment was inserted into the multi-clone sites of the vector to construct a new plasmid, pEGFP/pdx-1. E.colli strain DH5? was transfected with the new recombinant plasmid to expand it. Plasmid DNA extracted from the expanded DH5? was identifed by cutting with Hind Ⅲ, BamHⅠ nuclease and by DNA sequencing. Identified plasmid DNA was transfected into mouse embryonic stem cell line MESPU13 by carrying with liposome. RESULTS: A 876 bp cDNA fragment was amplified from mouse pancreatic cDNA by PCR and it was inserted into the vector pEGFP-N1 correctly. The fragment was defined to be pdx-1 gene by nuclease digestion and DNA sequencing. Mouse embryonic stem cell line MESPU13 was transfected with the new recombinant plasmid DNA. The green fluorescent protein report gene and pdx-1 gene expressed in transfected mouse embryonic stem cells within 24 h. CONCLUSION: Mouse pdx-1 gene is cloned and its recombinant eukaryotic expression vector carrying green fluorescent protein is constructed successfully. It provides a useful tool for further research on the function of pdx-1.