| ObjectiveCardiovascular disease is the leading cause of human death,coronary heart disease accounted for about 50%,of which the incidence of heart failure,WHO predicted that in 2020 the world will have a total of 25 million people died of coronary heart disease myocardial infarction(AMI)in the world by the end of the year.After myocardial infarction,myocardial cell necrosis and apoptosis can reduce the number of myocardial cells,which may lead to heart failure and even death.In the United States,there are more than 7 million people who have survived after myocardial infarction,of which 4 million 900 thousand have different degrees of heart failure.Although the treatment of coronary heart disease continues to improve,but 20% of patients diagnosed with myocardial infarction will die within 1 years.Therefore,it is urgent to improve the therapeutic effect of coronary heart disease.Various studies have shown that mi R-499-5p has a very close relationship with cardiovascular disease,especially in the development of coronary artery disease,vascular endothelial injury,mi R-499-5p expression level increased significantly.Therefore the separation of primary cell technology through this study,human umbilical vein endothelial cells as the research materials,focuses on the study of the mi R-499-5p with human umbilical vein endothelial cell inflammation,correlation of mi R-499-5p with human umbilical vein endothelial cell proliferation and apoptosis,mi R-499-5p expression and the correlation between the distribution of TCM syndromes in patients with coronary heart disease.Correlation.Methods 1 Isolation,Purification and Identification of Primary Human Umbilical Vein Endothelial Cells1.1 to non-infected,no deformed neonatal umbilical cord as a material,cleaning for enzyme treatment,collection of isolated cells,its cleaning and culture and passage;1.2 The cells were purified by differential adherence method,and the purified cells were screened and cultured.1.3 The expression of Ⅷ factor and CD31 protein was identified by cell immunofluorescence staining,and the cells were identified.1.4 MTT technique was used to map the cell curve of human umbilical vein endothelial cells and explore the effect of FBS concentration gradient on cell proliferation.2 Establishment of inflammatory vascular endothelial cell model2.1 Inflammatory stimulation of human umbilical vein endothelial cells by TNF-α inflammatory factor stimulation was used to establish inflammatory vascular endothelial cell model.2.2 MTT method was used to detect the difference of inflammation degree between model group and control group.2.3 MTT technique was used to map the cell curves of human umbilical vein endothelial cells and explore the effect of inflammatory reaction on cell proliferation.3 Different expression of mi R-499-5p in vascular endothelial cells with different inflammatory response3.1 Human umbilical vein endothelial cells were divided into control group and inflammatory group,the control group cells were treated with PBS blank human umbilical vein endothelial cells,and the inflammatory group was highly inflamed human umbilical vein endothelial cell model.3.2 total RNA extraction,reverse transcription technique for reverse transcription of cell RNA;3.3 The expression of mi R-499-5p in human umbilical vein endothelial cells was detected by real-time quantitative PCR.4 Different expression of mi R-499-5p on vascular endothelial cell prolife ration4.1 mi R-499-5p mimic in vitro mi RNA mimetic technique was used to establish the mi R-499-5p overexpression cell line by increasing the expression level of mi R-499-5p in human cells.4.2 Using mi R-499-5p inhibitor in vitro mi RNA inhibitor technique,the expression level of mi R-499-5p in human cells was down-regulated,and mi R-499-5p low expression cell group was established.4.3 The expression of mi R-499-5p in different groups was detected by reverse transcription and real-time quantitative PCR.4.4 MTT technique was used to construct the cell curve of human umbilical vein endothelial cells and explore the effect of mi R-499-5p expression on cell proliferation.4.5 RTCA cell real-time monitoring system detection technique was used to investigate the effect of mi R-499-5p expression on cell growth.4.6 To investigate the effect of mi R-499-5p expression on cell apoptosis by flow cytometry(FCM)4.7 DAPI staining was used to investigate the effect of mi R-499-5p expression on apoptosis.5 MiR-499-5p regulates SOX6 protein-mediated inflammatory response5.1 information biology technology and RNA library technology,screening and mi R-499-5p with anastomotic related protein;5.2 Using mi R-499-5p mimic in vitro mi RNA mimetic technique,the expression level of mi R-499-5p was up-regulated,and mi R-499-5p overexpressed cell group was established.5.3 Using mi R-499-5p inhibitor in vitro mi RNA inhibitor technique,the expression level of mi R-499-5p in human cells was down-regulated,and the mi R-499-5p low expression cell group was established.5.4 The expression of mi R-499-5p in different groups was detected by reverse transcription and real-time quantitative PCR.5.5 The expression of SOX6 protein in mi R-499-5p cells was detected by immunoblotting technique.The expression of mi R-499-5p and SOX6 protein was investigated.6 Study on the correlation between 6.MiR-499-5p expression level and TCM Syndrome Types6.1 cases: in 2016 1 to January 2017 in the First Affiliated Hospital of Guangzhou University of Chinese Medicine from inpatient department of cardiology patients as the research object,the number of patients included 180 cases of coronary heart disease,were consistent with the diagnostic criteria of ischemic heart disease,90 cases were male,90 female;the average age(49.6 + 15.5)years;the average duration(6.3.1.7)years,were excluded from the serious liver and kidney and hematopoietic system diseases such as primary disease,cancer,chronic infection,cerebrovascular disease,recent surgery and trauma,severe neurosis,psychosis;exclusion of TCM type two and see and no secondary type complex syndrome.Another 30 healthy people in the control group were excluded coronary heart disease patients,male,female,with a mean age of 15(47.6 + 12.5)years old.There was no significant difference in gender and age between the two groups.6.2 coronary heart disease patients in 2012 in accordance with the "traditional Chinese medicine and natural medicine treatment of angina pectoris of coronary heart disease clinical research technique" guiding principles for TCM syndrome;blood stasis type in 87 cases,male 45 cases,female 42 cases,average age(54.5 + 11.8)years old,the average duration of(5.5 + 2.1)years;phlegm heart vein type 21 cases,male 9 cases,female 12 cases,average age(49.5 + 9.8)years old,the average duration of(6.5 + 1.1)years;45 cases of qi deficiency and blood stasis,24 cases were male,21 female;the average age(52.5 + 9.8)years old,the average duration of(4.5 + 1.1)years;the heart and kidney in 27 cases,male 12 cases,female 15 cases,average age(51.5 + 9.8)years old,the average duration of(6.5 + 1.1)years.0 cases of qi stagnation and blood stasis,stagnation of Yin cold type in 0 cases,0 cases of Qi Yin deficiency,Yang deficiency type 0 cases,another 30 cases of healthy volunteers to exclude coronary heart disease.There was no significant difference in the general data and the factors that might influence the concentration of mi R-499(smoking,hypertension and heart failure).6.3 the expression level of mi R-499-5p in patients with coronary heart disease(CHD)and normal control group was detected by reverse transcription and real-time quantitative PCR;7 Statistical methodusing Alphaview SA 331 software for exposure,image photography,grayscale access;SPSS20.0 software package for statistical analysis;The measurement data were expressed as mean ± standard deviation(± S),all data were satisfied with homogeneity of variance,P > 0.05;Using variance analysis for comparing the data of two groups,P <0.05 showed a statistical difference;multiple comparison using two pairs of contrast method;significance level α take 0.05.Conclution 1 Establishment of inflammatory vascular endothelial cell model1.1 Inflammatory cytokines of human umbilical vein endothelial cells were stimulated by TNF-α inflammatory factor stimulation.After the establishment of inflammatory vascular endothelial cell model,MTT assay was used to detect the difference of inflammation degree between model group and control group.The results showed that TNF-Inflammatory factors stimulated the level of cell inflammation increased significantly;1.2 MTT technique was used to construct the cell curve of human umbilical vein endothelial cells and explore the effect of inflammatory reaction on cell proliferation.The results showed that the proliferation rate of TNF-α stimulated cells was lower than that of control group.2 Different expression of mi R-499-5p in vascular endothelial cells with different inflammatory response2.1 Human umbilical vein endothelial cells were divided into control group and inflammatory group,the control group cells were treated with PBS blank human umbilical vein endothelial cells,and the inflammatory group was highly inflamed human umbilical vein endothelial cell model.2.1 Compared with the normal control group,the expression of mi R-499-5p was up-regulated in the cells with high inflammatory response.3 Different expression of mi R-499-5p on vascular endothelial cell proliferation3.1 mi R-499-5p mimic in vitro mi RNA mimetic technique was used to increase the expression level of mi R-499-5p in cells,and mi R-499-5p cells were established.Q-RT-PCR showed that the model High expression of mi R-499-5p;3.2 mi R-499-5p inhibitor was used to down-regulate the expression of mi R-499-5p in mi R-499-5p inhibitor,and the mi R-499-5p low expression cell group was established.The Q-RT-PCR assay showed that the model Low expression of mi R-499-5p;3.3 MTT technique was used to construct the cell lines of human umbilical vein endothelial cells.The results showed that the cell proliferation of the cells with high mi R-499-5p expression was decreased compared with the normal control group.The expression of low mi R-499-5p cells Increased level of proliferation;3.4 Real-time monitoring system of RTCA cells was used to detect the cell proliferation rate of the cells with high mi R-499-5p expression compared with the normal control group.The cell proliferation rate of the low mi R-499-5p expression group increased;3.5 Using flow cytometry,the apoptotic rate of cells with high mi R-499-5p expression was decreased compared with that of normal control group.The apoptosis rate of cells with low mi R-499-5p expression was decreased.3.6 DAPI staining showed that the apoptosis rate of cells with high mi R-499-5p expression was increased compared with that of normal control group.The apoptosis rate of cells with low mi R-499-5p expression was decreased.4 MiR-499-5p regulates SOX6 protein-mediated inflammatory response4.1 Information biology technology and RNA library technology,screening with mi R-499-5p with anastomotic related protein,the results show that SOX6 protein promoter sequence with mi R-499-5p similar to the base sequence,suggesting that SOX6 protein may Controlled by mi R-499-5p;4.2 mi R-499-5p mimic in vitro mi RNA mimetic technique was used to establish the mi R-499-5p expression level in the cell,and the mi R-499-5p high expression cell group was established.Q-RT-PCR showed that the model High expression of mi R-499-5p;4.3 mi R-499-5p inhibitor was used to down-regulate the expression of mi R-499-5p in mi R-499-5p inhibitor,and the mi R-499-5p low expression cell group was established.Q-RT-PCR showed that the model Low expression of mi R-499-5p;4.4 Compared with the normal control group,the expression of SOX6 protein was up-regulated in mi R-499-5p cells and the expression of SOX6 protein was down-regulated in mi R-499-5p cells.5 Study on the correlation between expression level of MiR-499-5p and TCM syndromes of patients5.1 In this study,the distribution of TCM syndromes in patients with coronary heart disease was blood stasis type,phlegm-shaped heart vein type,Qi deficiency and blood stasis type and heart and kidney type.(P <0.05).5.2 The expression of mi R-499 in different groups was statistically significant,and the expression level of mi R-499 was significantly higher than that of blank control group(P<0.05)(P <0.05).The content of mi R-499 in the blood stasis type was higher than that in the control group(P <0.05).Conclusion1 Inflammation can lead to the decline of human umbilical vein endothelial cell proliferation rate,increased apoptosis rate,suggesting that inflammatory injury is an important cause of vascular endothelial injury;2 Inflammation can significantly increase the expression of mi R-499-5p in human umbilical vein endothelial cells,suggesting that the intracellular expression of mi R-499-5p is related to inflammation.3 The high expression of MiR-499-5p can significantly reduce the cell proliferation rate of human umbilical vein endothelial cells and affect the cell growth.In contrast,the low expression of MiR-499-5p can improve the cell proliferation rate of human umbilical vein endothelial cells Low cell apoptosis rate;... |
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