| [Objective]:Deep vein thrombosis (DVT) is the most common surgical complication, often resulting in serious consequences, and its mechanism is complex and not completely known, which need for further exploration. It is well known that vascular endothelial cells (VEC) Apoptosis plays an important role in the pathogenesis of DVT, which may be regulated by microRNAs. MicroRNAs are widely involved in regulation of cell differentiation, proliferation, physiology and pathophysiology of apoptosis, and miR-126 are closely related with the VEC and vascular function. In addition, Bcl-2, Bad were found differentially expressed during cell apoptosis process. In this study, we tried to study the mechanism of miR-126 in DVT in vivo and in vitro by gene silencing/ overexpression way. HE staining, TUNEL Apoptosis Detection test were used to observe the effects of miR-126 in DVT formation and the apoptosis of endothelial cells. Bcl-2, Bad protein expression were also tested by Western bolt analysis in different groups, to explore the relevance of miR-126 and Bcl-2, Bad in DVT and provide a new methods for further clinical treatment.[Materials and methods]:1. groups and modeling:the 72 SD rats (250 ± 20 g), male and female, irrespective of age, adaptive feeding 4 Day, were randomly divided into four groups.A.The normal control group (12):A11 rats in this group were subjected to intraperitoneal injection of 20ml saline.B. DVT model group (20):All rats in this group were subjected to intraperitoneal injection of 20ml saline. Then ventral midline laparotomy were carried out to expose inferior vena cava, which were sutured by 4-0 Ethicon suture ligation.After operation all rats were feed normally.C. miRNA-126 silent group (20):All rats in this group were subjected to intraperitoneal injection of compound 400 pmol antagomir of 20ml,to silence the expression of miR-126 gene expression, Four days after the injection, The operation process were carried out as mentioned above.D. miRNA-126 expression group (12):All rats in this group were subjected to intraperitoneal i injection of compound 400 pmol antagomir of 20ml.Four days after the injection, the operation process were carried out as mentioned above.the vein wall acquisition and pathology.2. Specimen collection and pathologic examination of the vein wallAccording to the results of preliminary experiments, random sample half of the group below after modeled in 2h and 24h, control group, DVT model group, miRNA-126 silent group, miRNA-126 overexpression group. After anesthesia and disinfection, open the abd -ominal cavity of the mice, cut off the inferior vena cava and its contents between the lgature to 2 cm away from the bifurcation of the common iliac vein, then observed whether thrombus exist or not in perusal, and put partial fresh tissue into 4% paraformal-dehyde fixed paraffin sections and observed the thrombosis state, the rest part is flushed by saline, put the Specimen of the vein wall into the frozen tube after removing the thrombus and preserve in the nitrogen canister.3. Using the TUNEL staining vein endothelial cell apoptosisTake the organizations at different time points from the nitrogen canister, then make the parafin section and in accordance with TUNEL cells Apoptosis detection kit (Shanghai Yi San) Manual operation detects the occurrence of venous endothelial cell apoptosis, observing and photographing under a fluorescence microscope after DAPI stained.4. Western bolt to detect Bcl-2, Bad protein expression in SD rats DVT vein wall tissueA. Extracted total protein of different time site stored in liquid nitrogen in each group of the vein wall material by using tissue lysates, then test total protein concentration by using the BCA method.B.After preparing the SDS-PAGE electrophoresis, finish transfer membrane, blocking, antibody incubation and other operations, using the ChemiScop Series 3600 chemiluminescence imaging system camera to display the results, and with Chemi Analysis Analysis to calculated the gray value.C. use SPSS17.0 statistical package for the data processing, each set of values with the mean ± standard deviation (x ± s) that the groups were compared using ANOVA (One-Way ANOVA); each set of results with P<0.05 it was considered statistically significant.[Results]1. visual observation of thrombosis2h sites, DVT rats, silent group and high expression seen with the naked eye view vein wall edema, color burn, no significant thrombosis; 24h sites in addition to the normal group, the other three significant vein wall edema, swelling of the inferior vena cava and bruising obviously, there are a lot of thrombosis, and DVT model group and miRNA-126 silent group the most obvious..2. HE staining thrombosis2h site thrombosis less visible at 24h, DVT group and miRNA-126 is completely silent group thrombosis, miRNA-126 high expression of a large number of thrombosis.Except to the normal group, the other three in 2h,24h sites under HE staining are visible thrombosis; at 2h site thrombosis is fleabite, at 24h. DVT group and miRNA-126 silent group are completely thrombosis. miRNA-126 high expression group present a large number of thrombosis.3. Tunel Apoptosis testat different time sites, by observing the fluorescence intensity (the degree of apoptosis) to determine apoptosis; at 2h sites, silence miR-126 group situation show more severe apoptosis than DVT model group, and normal group and high expression of miR-126 group did not see significant apoptosis; at 24h sites, in addition to the normal group were seen apoptosis occur, compared with the DVT model group, miR-126 silence group show a severe apoptosis, and miR-126 overexpression group, the degree of apoptosis compared with the model group DVT is lighter.4.Western bolt to detect Bcl-2, Bad proteinCompared with normal group, the group of Bcl-2, Bad sites at different times have changed. Combined with statistical analysis,2h sites are not all equal in the Bcl-2 protein group (F= 2982.881, p<0.001), there are significant differences in the 24h sites are not all equal in the Bcl-2 group (F=352.747, p<0.001), statistically significant, and in 2h, 24h sites, DVT group with high expression of miRNA, miRNA and miRNA silencing group expression group was significantly (P<0.001); at 2h site, Bad proteome inside are not all equal (F=571.01, p<0.001), there are significant differences in the 24h sites are not all equal in the Bad group (F=393.684, p<0.001), statistically significant, and in 2h, 24h sites, DVT group and the group of miRNA expression, miRNA and miRNA silencing group expression group was significantly (P<0.001)[In conclusion]1、miR-126 and DVT formation and vein endothelial cell apoptosis were negatively correlated2、miR-126 and apoptosis related protein Bcl-2 was positively correlated negatively correlated with Bad... |
PDF Full Text Request