| ObjectiveWe built the recombinant adrenomedullin by combined the aderenomedullin gene with the eukaryotic expression vecotr. Then direct injected the different dose of recombinant plasmid DNA into the gastrocnemius of rat to build the exhausted exercise model. We evaluated the protective effect of the recombinant adrenomedullin and dosageeffect in the myocardium tissue and compared with the protective effect of reduced glutathion.Methods:1. Build the recombinant gene of adrenomedullin (pVAXl-ADM): First of all, we extract total RNA of adrenal gland from male SD rats to get the cDNA of ADM gene by processing RT-PCR. Once we collected enough cDNA models, we cloned these into PMD-18T vector. Then we sub cloned the cloning item into eukaryotic expression vecotr pVAX1to get the final outcome as pVAX1-ADM. Later, we double collected large amount of recombined plasmid that contains enough concerntration and purity by using alkaline lysis method and polyethylene glycol method.2. We separated18male SD rats of those each weights around (203±18.4) g into3groups randomly, each group had6rats. These groups were set for different uses:control group, pVAX1group, and pVAX1-ADM700μg/kg group. We injected lml normal saline, pVAX14plasmid normal saline solution1ml and700μg/kg pVAX1-ADM normal saline solution lml separately, we injected them once a week, and the experiment lasted for4weeks. When the model has been successfully built, we used immunohistochemical method to examine the outcome from recombined plasimid pVAX1-ADM of rat’s myocardium tissue.3. Studying the protective effect of exhausted exercise rat on the hypoxia heart with different volumes of pVAX1-ADM:We assigned48male SD rats of those each weights around (223.24±23.77) g into6groups randomly by their weights, each group had8rats. The groups were set for different uses as follows:control group (C)、exhausted group (Ex)、GSH group (G), pVAX1-ADM700μg/kg group (P1)、pVAX1-ADM1100μg/kg group (P2), and pVAXl-ADM1400μg/kg group (P3).We forced the rats to have activities according to models of their exhausted-induced hypoxic heart for one time. And we had this experiment for all groups of rats except control group. We injected lml normal saline with group C and group Ex, we also injected0.125g/ml GSH normal saline solution lml with group G once a week. At the same time, we injected1ml of each pVAX1-ADM700μg/kg、1100μg/kg and1400μg/kg with group P1、P2and P3once a week. On the final week of the experiment, right after the rats got exhausted through activity, we collected their myocardium tissue to determine the level of MDA, SOD, CK and LDH. We have also done studies on tectology of myocardium tissue. At the end, we collected blood samples from the rats to detect several index such as plasma viscosity, erythrocyte aggregation index, RBC deformability index and so on.Results:1. The sequence of the recombined plasmid DNA we built has been proven to be consistently with the ADM gene we found in Genebank. We also found a positive outcome by examining double digestion by XbaⅠand HindⅢ And we used UV scenery photometric method found purities of pVAX1-ADM which has been purified as: OD260-0.924,OD280-0.505, OD260/OD280=1.828, concentration as4.63mg/ml.2. After studying of tetology of myocardium tissue of rat, it suggested that the ADM expression level in gastrocnemius was lower in control group and pVAXl plasmid group whereas high in pVAXl-ADM700μg/kg group.3. The final results of determining MDA, T-SOD, CK and LDH of myocardium tissue suggested that:compare to group Ex, the contents of both LDH and MDA for group P1, P2, P3, and group G decreased apparently (P<0.05), the result has significant of different(P<0.05), has statistical significant; And the content of T-SOD has increased apparently (P<0.05), the result has significant of different(P<0.05), has statistical significant. The examination of tectology suggested that there was a severe damage on group Ex’s structure of myocardium tissue, inflammatory cells gathered, edema obviously. In addition, Compare to group Ex, the damage of structure of myocardium tissue for group P1, P2, P3and group G have been each alleviated apparently, a relief of tissue edema has also been showed. The result of hemodynamics showed that the high shear viscosity of whole blood, low shear viscosity of whole blood, Plasma viscosity, erythrocyte aggregation index, RBC deformability index of group PI, P2, P3and G were lower than the group Ex (P<0.05), the result has significant of different (P<0.05), has statistical significant; the hematocrit of group P1, P2, P3and G were higher than the group Ex (P<0.05), the result has significant of different (P<0.05), has statistical significant. Conclusions:1. We built pVAXI-ADM of rats successfully, the purity and concentration of DNA we extracted from the examination were qualify for the following up examination.2. The ADM expression was upregulated in gastrocnemius of rat by the induction of700μg/kg pVAXI-ADM, that suggested that the experimental model was succeed, and the recombined plasmid could uptake by the tissue and expressed in the tissue.3. The induction of700μg/kg、1100μg/kg、1400μg/kg pVAX1-ADM and GSH may increase the T-SODactivity, but decrease the contents of CK, LDH and MDA, improve the hemodynamics and the damage by hypoxia induced, to finally protect the heart against exhausted-induced hypoxic damage and have the dose effect. The optimal protective effect is1400μg/kg pVAX1-ADM... |
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