| Endpthelial cells regulate tone of blood vessel through the release of vasoactive substances. With the increasing of age , the function of endothelium demage, but mechanisms are unclear. Endothli - um can release Endothlium - derived relaxing factor ( EDRF) , which causes the smooth muscle cells to relax. Nitric oxide (NO)has been a candidate for this substance. Increas - ing age can affect endothelium function, in which NO plays a very important role. It will be important significance to investigate the action of NO.At present , the results of study are not identical on the change of NO in aging . In our experiment ,it is be use drugs,such as: Ach,NG - nitro - L - arginine( L - NNA) ,L - arginine( L - Arg) to determinate endothelium - dependent relaxtion of aorta ring in different age groups, which observe the effect of aging on relaxation and contraction reponse of endothelium . At the same time, the study used in immuno-cytochemistry to observe the expression of inducible NOS (iNOS) and enduthila NOS(eNOS) in different age groups,so that it may be much directly and much precisely to reflact the change of NO, aim at to find out the mechanism on NO/NOS system and occurenc, development of aging.Metarilas1. Animals: Wistar old rat (n =20) , adult rat(n =20)2. Reagent; L - NNA,L - Arg,NE,Ach,SABC immunocytochemi-cal test kits,iNOS test kits,eNOS test kits.3. Apparatus; tissue - organ water bath system, auto - equilibrium recording meter of desk type, acidometer, microtome, light microscope , figure disposal meter. , Imaging MetaMorphous SystemMethods1. Isolated animal experimentWister rat ,4 - 5 mo old ( adult group n = 10) ,20 - 22 mo old ( old group n = 10) were anesthetized with phenobarbital sodium (60mg/kg). The rats were then decapitated and the chest and abdomen were opend . Subsequently the aorta were excised and placed in cold (4℃) kreb' solution isolated vessels were cleand of adhering tissue and cut into rings of 3 - 4mm in length. Aortic rings were mounted horizontally between two stirrups in organ chambers filled with 20ml kreb' solution (37℃, with a gas mixture of 95% O2and 5% CO2) . One trirrup was connected to an anchor and other to a force transducer for recording of isometric tension . After a 30 min equilibration period ,vessels were contracted with 2 x 10-7 mol/L NE,and then adding Ach,L - NNA,L - Arg and so on ,to record the changes of tension of vessels ring.2. Immunocytochemistry experimentWister rat ,4 - 5mo old ( adult group n = 10) ,20 - 22 mo old (old group n = 10) were anesthetized with phenobarbital sodium (60mg/kg). The rats were then decapitated and the chest and abdomen were opend .Aorta were dissected for 2cm and embedded with parafifin , sections of 5 m were made.Immunocytochemical staining of serial sections was done according to SABC techniques . Brown particals of positive staining were seen in section of histiooytic plasm . Then the sampies were observed nuder light microscope. The Imaging MetaMorphous System were used to detect the gray value of iNOS and eNOS.2.3 Statistical analysisResults are expressed as mean s. Statistical comparisons between groups were performed by the t - test. Values of P < 0. 05 were considered significant.Result1. Changes of isolated vessels tension with drugs.1. 1 Isolated aortic rings of rat of intact endothelium were contracted with 2 x 10-7 mol/L NE, the contractile result of two groups did not significant differention, and all can maintenance on contracted behavior.1. 2 After contracted with NE, cumulatively increasing concentrations of Ach (10-8 ~ 10-4 mol/L) were added to steady - state NE induced contractions. The result shows the Ach - induced relaxation in two groups rat was endothelium - dependent. Ach - dependent relaxation in old rat was significant decrease compare to that of adult rat(P<0.01).1. 3 After pretreatment 20min with L - NNA( 10-4 mol/L) to inhibit NOS, isolated aortic rings of old and adult rat can not be induced endothelium - dependent relaxtion by Ach. |
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