| Objective: Master CMVECs primitive culture techniques,and preparation for multiple sets of lipopolysaccharide damage model of rat cardiac microvascular endothelial cells,observe the cell morphology by microscope,then providing the morphological basis for subsequent experiments.To investigate the effects for the pretreatment of Ulinastatin on inflammatory response to LPS induced damage to the myocardial microvascular endothelial cells.To investigate the effects for the pretreatment of Ulinastatin on response to LPS induced damage to the apoptosis of microvascular endothelial cells.To explore the protective mechanism of ustantidine on myocardial injury in sepsis rats,and provide a theoretical basis for the treatment of myocardial injury of sepsis rats in the early stage.Materials and Methods: Choose the myocardial tissue of rats,clean the blood with PBS solution,and to cultivate the rat heart cells until it grow into the cell monolayer,choose the third generation of endothelial cells,after 0.25% trypsin digestion then the cells had been divided into 6 groups: 1): the blank control group: join serum free medium only;2)Model group 1: adding a non-serum medium that contains 0.01 ?g/ml LPS.3)Model group 2: adding a non-serum medium that contains 0.1 ?g/ml LPS.4)Model group 3: adding a non-serum medium that contains 1 ?g/ml LPS.5)Model group 4: adding a non-serum medium that contains 10 ?g/ml LPS.6)Model group 5: adding a non-serum medium that contains 100 ?g/ml LPS.The morphological changes of endothelial cells for the incubation and incubation of 24 h were studied under the inverted microscope.Then To continue the intervention of later cells: 1)the control group did not do any treatment;2)the pretreatment group of atto(10000u/ml);3)low concentration UTI(5000u/ml)pretreatment group;4)light concentration UTI(10000u/ml)pretreatment group;5)high concentration UTI(20000u/ml)pretreatment group.By immunohistochemistry and Western blot detection methods to detect the inflammatory factors for the cells,specific indicators including interleukins(IL-1),IL-6,IL-8,tumor necrosis factor-?(TNF-?),the expression of cell autophagy related protein Atg5,Beclinl and LC in the cells;Later,the data were summarized and analyzed.The Western blot method was used to detect changes in the expression levels of Bcl-2 and Bax protein in each group of endothelial cells.MTT tests to detect cell activity in each group.Flow technique was used for the rate of apoptosis in each group.Healthy and clean male Wistar rats were randomly divided into 6 groups and each for 10 rats.Before the preoperative fasting 12 hours,the rat model of sepsis was established,and the specific experimental group were as follows: control group,the rats did not do any treatment;Sham operation group: postoperative 0.9 physiological saline of the waiting volume;Atorvastatin group(50000U/kg);Low ulinastatin group(20000 u/kg),middle ulinastatin group(50000 u/kg),high ulinastatin group(100000 u/kg): after postoperative 24 hours,then to take the artery of rats and separation of left ventricular myocardial cells to observe the specific changes of cells;At the same time to take the ELISA method in the rat serum myocardial calcium,protein and tumor necrosis factor I and TNF-?;Radiation immune treatment did related tests for the rats myocardial endothelin-1 and corresponding myocardial silk crack protein kinase,later do the analysis of the experimental data.Results: As the concentration of LPS increases,the level of endothelial injury in the rat cells is progressively greater.The light microscope showed that with the increase of the LPS concentration,the endothelial cells were gradually becoming multilateral or even flat.At the same time,we observed that the interstitial space between the small vessels increased significantly,decreased the number of cells,and arranged the disorder.The scanning electron microscopy results showed that the endothelial cells were hollow and the villi were atrophied and distorted.Transmission electron microscopy(sem)showed that with the increase of concentration of LPS,the nucleus pycnosis gradually,highly expanding golgi complex,mitochondria lesion in vacuoles,cell fibrous lipoprotein precipitation phenomenon occurring at the same time;This phenomenon becomes more and more serious as the concentration of LPS increased.IL-1?IL-6?IL-8 level comparison: in High UTI grou P,Middle UTI grouP,Low UTI group,the content rises continuously,the P value of each group was less than 0.05,the difference was statistically significant;TNF-? level comparison: in High UTI group(300.87±12.89ng/ml),Middle UTI group(300.87±12.89ng/ml),Low UTI group(430.26±12.11ng/ml),Atorvastatin group(500.99±14.33ng/ml)and Control group(658.88±15.37ng/ml),the content rises continuously,the P value of each group was less than 0.05,the P value was 0.000,0.001,0.029,0.036 ? 0.049,the difference was statistically significant;Autophagy related protein IL-1? and LC3-1expression level: in High UTI group,Middle UTI group,Low UTI group,Atorvastatin group and Control group,the content rises continuously,the P value of each group was less than 0.05,the difference was statistically significant.Cell apoptosis rate comparison: in High UTI group(0.18±0.08,0.23%),middle UTI group(0.35±0.13,0.59%),low UTI group(0.45±0.13,0.72%),actora tachin group(0.78±0.19,1.13%)and control group(2.23±0.23,4.56%),the content rises continuously,the P value of each group was less than 0.05,the P value were 0.003,0.048,0.030,0.039 ? 0.000,the difference was statistically significant;Endothelial cells level of Bcl-2 protein comparison: in the control group,atorvastatin group,low UTI group,middle UTI group,high UTI group;the content rises continuously,the P value of each group was less than 0.05,the difference was statistically significant;Bax protein content comparsion: In high UTI group,middle UTI group,low UTI group,atorvastatin group and control group;the content rises continuously,the P value of each group was less than 0.05,the difference was statistically significant.Myocardial protein cTnl expression level analysis: in Sham-operated group(1.00±0.56ng/ml),High UTI group(1.23±0.23ng/ml),Middle UTI group(2.56± 0.38ng/ml),Low UTI group(3.03±0.58ng/ml),Atorvastatin group(4.33±0.46ng/ml)and control group(6.98±0.39ng/ml),the content rises continuously,the P value of each group was less than 0.05,the difference was statistically significant;TNF-? concentration analysis: in Sham-operated group(211.87±35.48pg/g),High UTI group(238.23± 30.23pg/g),Middle UTI group(273.55±51.04pg/g),Low UTI group(345.88± 30.88ng/ml),Atorvastatin group(457.34±45.27ng/ml)and control group(766.78± 43.89ng/ml),the content rises continuously,the P value of each group was less than 0.05,the difference was statistically significant;ET-1 expression level analysis: in Shamoperated group(168.34±27.71pg/g),High UTI group(170.44±28.79pg/g),Middle UTI group(234.34±29.33pg/g),Low UTI group(278.99±28.34ng/ml),Atorvastatin group(344.62±27.85ng/ml)and control group(677.32±38.74ng/ml)the content rises continuously,the P value of each group was less than 0.05;the P value were 0.000,0.000,0.003,0.001,0.025 and 0.003,the difference was statistically significant.Conclusion: Lipopolysaccharides could cause the internal damage to the heart cells of the rat,and the degree of damage to the LPS concentration were positively correlated.Substantial effects were presented for the UTI to inhibie the LPS induced damage within the myocardial microvascular endothelial cells.The process of the apoptosis of inflammatory cells in the myocardial microvascular endothelium that can effectively promote LPS induced cells.Ustodin could effectively reduce the levels of myocardial protein in the rats with sepsis,reduce the incidence of inflammatory response,which is deserve to be recommended clinically. |
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