The Study Of The Effect Of Collagen, Skeletin And Na～+/H～+ Exchange Inhibitor On Myocardiopathy Of Diabetes

ObjectiveThis study investigated the content changes of myocardial collagen and skeletin at different stages of DM rats. So as to illustrate which role they played in myocardial changes of DM and the effect of Na+/H+ exchanging(NHE) inhibitors on calcium overload in myocardial cells of DM.Methods1. Cardiac muscle model of DM rats36 SD rats with the weight of 250-300g were selected and among them 18 ones were injected with streptozotocin (60mg. Kg-1 ,i. p. , Sigma company). So the model of DM rats were established with blood glucose over 16.7mmol/l after seven days. All of them were divided into six groups at random with six in each group. That is : DM1 (DM model rats treated with 1.5% pentobarbital intrap-eritoneally in the 1st month) , DM3 ( DM model rats treated as that in DM1 but in the 3rd months, DM6, C1(control group 1 killed at the same time as DM1), C3 and C6.2. collagen contents: The collagen contents of myocardial muscle were determined by the hydroxyproline assay. The hydroxy proline contents were measured with the chloramine T method, which multiply 7. 46 to obtardial collagen contents.3. Myocardial immune histochemical stain;This measure was used to determine collagen I, Ⅲ,TGF -β1 and actin. The procedures were as follows :(1) slides detachment preventing management(2)slices deparaffin(3) endogenous enzyme inactivation(4) thermal repairment of artigen(5) adding Caprine serum sealing liquid s at room temperature for 20min.(6) Adding 1:100 diluted antibody( mice or rabbit IgG) to stay overnight at 4 ℃ , washed by PBS for 2min and 3times.(7) Adding biotinized IgG of rabbit - anti - mice. Stored at 20 ℃#- 37℃ for 20min and washed by PBS for 2min and 3times.(8)SABC added, stored at 20 ℃-37℃ for 20min and washed by PBS for 5min and 4times(9)DAB coloration (10) Hematine staining4. Collagen I ,Ⅲ, TGF -β1intergrated optical density analysis c(by microscopic image analyzer Meta Morph/DplO/BX51)5. Specimen for light microscope preparationThe myocardial specimen with 4um section was embedded in paraffin and stained by MASSON then observed with light microscope(LM).6. Specimen for TEM preparationThe specimen was fixed in osmate, washed by PBS for 5min and 4 times and then dehy cbated by alcohol and acetone in gradient separately, next embedded in epoxy resin, and aggregated and sliced, at last observed and photographed with TEM.7. Culture of myocardial cells of DM ratsEighteen rats of DM were obtained among 100 rats (10g-22g) at the age of five days just as stated above. These experimantal rats were anesthe tized with 1.5% pentobanbital (5mg.Kg-1, i, p. ). The cardiac tissues were excised under sterile conditions and cut in 1 - 2mm . These tissues were then digested by 0.125% trypsin and eentrifuged. The cardial muscle cells collected were re suspended with D MEM medium containing penicillin and streptomycin at PH 7. 4 at 31℃ for 90min in incubator and then inoculated into culture flask of 50ml. Single layer cells were selected after four daysCulture for experiment8. I/R injury model preparation and groupingSeveral culture flask covered with a single layer cells at the bottom were assigned into from groups.(1) DM Group ( DM simple hypoxia group )(2) DM - EIPA Group ( Adminstration in whole course of hypoxia/reoxida-tion )(3) DM - EIPA Group 1 ( Administration before reoxidation group 1)(4)DM -EIPA Group2 (Administration before reoxidation group2)9. Measurement of intracellular calcium and surface of single cell Myocar-dial cells were stored in medium containing 2. 5 mmol/L Fura -2 Am at 37℃ for 3min and washed with medium for 2 or 3 times, then incubated for 30min. These cells then could be measured with inverted Fluorescence Microscope Intracellular calcium was determined from Cynkiewicz formula: [ Ca2+] : = Kd ( FD/ Fs) (Fo-FMmin)10. Statistics ManagementsAll the data were expressed x+s s and SPPS 11.0 soft ware was used and One -way ANOVA, x2 test were taken at P <0.05.R...