| Objective:1. To construct rat adrenomedullin eukaryotic expression vector pVAX1-ADM.2. To explore the effect and mechanism of recombinant adrenomedullin in Ischemia-reperfusion of rat abdominal flaps.Methods:3. Rat adrenal total RNA was extracted and then ADM gene was obtained by RT-PCR method. ADM gene was cloned into the T-vector,and subcloned into the eukaryotic expression vector pVAX1 further.4. 30 male SD rats, weight 200±10g, according to the weight were randomly divided into 5 groups: empty plasmid group (pVAX1) , 200μg dose, 400μg, 600μg and 800μg dose recombinant plasmid (pVAX1-ADM) group. The five groups abdominal flaps were injected with recombinant plasmid for four weeks, the dose of recombinant plasmid respectively was 0μg, 200μg, 400μg, 600μg and 800μg, once a week. RT-PCR, western blot and immunohistochemical examination were utilized to determine the levels of recombinant plasmid transcription and protein expression in target flaps, to identify the optimal dose of recombinant plasmid.5. 32 male SD rats, weight 200±10g, according to the weight were randomly divided into 4 groups: control group, IR (ischemia reperfusion) model group, Positive medicine group (Verapamil group), Recombinant plasmid group. Before operation, rat in Recombinant plasmid group were injected with 600μg recombinant plasmid to their abdominal flaps and the other three groups were injected with the same capacity of normal saline, once a week. After 4 weeks, Ischemia-reperfusion models were set up, ischemia was allowed for 9 h, then reperfusion for 12 h. In operation, verapamil solution (2mg/kg) was injected to intraperitoneal in Verapamil group, the other three groups were injected with the same capacity of normal saline, 10 minutes before reperfusion. After operation, check the contents of MDA, SOD and ET-1 in target flaps, the contents of CK, LDH, and TNF-αin serum; and histomorphology examination of target flaps was performed at 7 days after surgery.Results: 1. Obtained ADM gene sequence was consistent with GeneBank rat ADM sequence. Identifying the eukaryotic expression plasmid by PCR XbaI,HindIII digestion,results showed the recombinant plasmid was present. Spectrophotometry was used to judge the recombinant plasmid , results was OD260/280=1.85±0.05. Recombinant plasmid concentration was 4.0 mg/mL.2. Detection of gene expression in rat abdominal flaps: results from semi-quantitative RT-PCR, western blot and immunohistochemistry experiments demonstrated that the ADM expression was increased.3. Compared with the IR model group, the content of MDA in pVAX1-ADM group in abdominal flaps decreased 38.50% (p <0.01), activity of SOD increased by 50.67% (p <0.05), ET-1 level decreased 54.73% (p <0.01); content of CK in serum decreased 53.84% (p <0.05), content of LDH reduced 29.18% (p <0.01), content of TNF-αdecreased by 53.89% (p <0.01). Additionally, Compared with the verapamil group, the content of MDA in ADM plasmid group in target flaps increased 8.61% (p > 0.05); activity of SOD increased 15.84% (p > 0.05), ET-1 level increased by 9.53% (p > 0.05); content of CK in serum increased 11.93% (p > 0.05), content of LDH increased 33.51% (p <0.05), content of TNF-αdecreased 18.19% (p >0.05). Compared with the IR model group, the structure of flaps in PVAX1-ADM group and the verapamil group showed that only a few infiltration of inflammatory cells occurred and interstitial edema decreased significantly.Conclusion:1. ADM expression gene was connected to the the eukaryotic expression vector successfully. Recombinant plasmid concentration is 4.0 mg/mL.2. Recombinant plasmid was injected to abdominal skin flaps which increase the expression of ADM. The 600μg dose recombinant plasmid group was selected as the best expression group.3. Rat target skin flaps ADM expression was enhanced which can reduce injury of the tissues and cells in the process of ischemia reperfusion. |
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