| Deep vein thrombosis is a multi-system,multi-factor,multi-gene involved disease involving venous endothelium,platelets,white blood cells,coagulation factors,fibrinolysis factors,for example,venous endothelial SIRT1,P-Selectin,PSGL-1,vWF Many gene expression changes such as TM,TF play an important role in the formation of DVT.Based on the establishment of the CVT/6 mouse DVT model,we screened and analyzed the venous endothelial thrombosis/non-formation related genes by high-throughput sequencing.It was found that compared with the NC group,the DVT group Mmp-12,the expression of Mmp-13 was up-regulated.Compared with the Blank group,Klf15 expression in Klf15-siRNA group was down-regulated,Mmp-12 and Mmp-13 expression were up-regulated,and thrombus wet weight was significantly increased.It has been reported that KLF15 competitively inhibits NF-?B signaling pathway during arterial thrombosis,which reduces vascular inflammation and inhibits thrombosis.It is also found that Mmp-12 and Mmp-13 can affect arterial thrombosis.Therefore,this subject aims to study:1.The effect of KLF15 on the expression of MMP-12 and MMP-13.2.The effect of Mmp-12 and Mmp-13 on deep vein thrombosis.3.Klfls affects the expression of Mmp-12 and Mmp-13 and the effect on deep vein thrombosis.[Objective]Human umbilical Vein Endothelial Cells(HUVECs)were cultured in vitro,and plasmids PCMV3-KLF 15-over transfected HUVECs.KLF15-siRNA fragments were transfected into HUVECs to interfere with the expression of KLF15 gene.Real-time PCR and Western Blot were used to evaluate the expressions of MMP-12 and MMP-13,to investigate whether KLF15 can regulate the expression of MMP-12 and MMP-13.2.Under the action of small animal anesthesia system(R510-25),the DVT model of C57BL/6 mice was constructed by inferior vena cava stenosis;Mmp-12-siRNA,Mmp-13-siRNA fragment were synthesized,and injected into tail veins.The expressions of Mmp-12 and Mmp-13 genes in mice were observed.The mortality,thrombolysis rate,inferior vena cava length and wet weight of DVT model of C57BL/6 mice were assecs after 24 hours.Real-time-PCR was used to evaluate the expressions of Mmp-12 and Mmp-13 in the DVT model.The effects of Mmp-12 and Mmp-13 on the formation of DVT in mice were investigated.[Materials and Methods]The first part:HUVECs were cultured,and plasmid PCMV3-KLF 15-over transfected HUVECs and KLF15-siRNA fragments were transfected into HUVECs to interfere with the expression of KLF15 gene,and the expression of downstream MMP-12 and MMP-13 was detected.1.The HUVECs were cultured in vitro,and the plasmid PCMV3-KLF 15-over was transfected into HUVECsKLF15-siRNA fragment transfection HUVECs interfered with the expression of KLF15 gene.The total RNA of HUVECs was isolated using TRIzol(invitrogen)reagent,and the RNA of HUVECs was reverse transcribed into cDNA by real-time PCR.The expression of MMP-12 and MMP-13 in HUVECs was detected by real-time PCR and Westerm Blot.GAPDH as an internal reference.Each quantitative RT-PCR sample was normalize to the expression of GAPHD.2.The experimental data were analyzed by SPSS 22.0 statistical software,and the effect of KLF15 on the expression of MMP-12 and MMP-13 was analyzed.The second part:The stenosis method was used to construct the deep vein thrombosis model of C57BL/6 mice.The effects of Mmp-12-siRNA and Mmp-13-siRNA on thrombosis in mice were observed.1.50 C57BL/6 mice were selected and raised in SPF animal room.There is no restriction on gender and their weight was about 24-26 g.They were randomly divided into 5 groups,10 in each group.NC group(group A,n=10),DVT group(group B n=10),blank group(group C,n=10),Mmp-12-siRNA group(n=10),Mmp-13-siRNA group(n=10).NC group and DVT group were not treated for 24 hours before modeling;Blank group(200 ul normal saline was injected into the tail vein 24 h before modeling;Mmp-12-siRNA group and Mmp-13-siRNA group were injected into the tail vein 24 hours before modeling).200 ul Mmp-12-siRNA,Mmp-13-siRNA.2,24 hours after the tail vein injection,the mouse inferior vena cava thrombosis model was established by the Infenor vena cava stenosis method.The thrombus and the vascular endothelium formed at the thrombus site were dissected 24h after modeling,and the thrombus related indicators were measured and real-time was used.The expression of Mmp-12 and Mmp-13 in venous endothelium was detected by PCR.3.The experimental data were analyzed by SPSS 22.0 statistical software.The effects of Mmp-12 and Mmp-13 on deep vein thrombosis in mice were analyzed.[Result]Experiment 1:HUVECs were cultured in vitro,and plasmid PCMV3-KLF 15-over transfected HUVECs and KLF15-siRNA were used to interfere with the ey pression of HUVECs gene,and the expression of downstream genes was detected.1.Real-time PCR and Western Blot were used to detect the expression changes of Mmp-12 and Mmp-13 in downstream HUVECs.According to the protein band analysis,the expression of KLF-15 in the PCMV3-KLF15-over group was significantly up-regulated compared with NC1;the expression of KLF-15 in the KLF15-siRNA group was significantly down-regulated compared with the NC2 group.Compared with NC1,MMP-12 was significantly down-regulated in the PCMV3-KLF 15-over group;MMP-12 expression was significantly up-regulated in the KLF15-siRNA group compared with the NC2 group.Compared with NCI.MPCM-13 was significantly down-regulated in the PCMV3-KLF 15-over group;MMP-13 expression was significantly up-regulated in the KLF15-siRNA group compared with the NC2 group.According to the statistical analysis,the expression of KLF15 protein in the PCMV3-KLF 15-over group was significantly higher than that in the NCI group(P<0.05).The expression of KLFl5 protein was significantly lower in the KLF15-siRNA group than in the NC2 group.Statistically significant(P<0.05).Compared with NC1 group,the expression of MMP-12 protein in PCMV3-KLF15-over group was not significantly different(P>0.05).The expression of MMP-12 protein was significantly increased in KLF15-siRNA group compared with NC2 group.Statistically significant(P<0.05).Compared with NCl group,the expression of MMP-13 protein in PCMV3-KLF 15-over group was significantly decreased(P<0.05).The expression of MMP-12 protein was significantly increased in KLF15-siRNA group compared with NC2 group.Academic significance(P<0.05).Experiment 2:The deep vein thrombosis model of C57BL/6 mice was constructed by stenosis method.The effects of Mmp-12-siRNA and Mmp-13-siRNA on thrombosis in mice were observed.(1)Anesthetic effect:During the anesthesia,different concentrations of isoflurane anesthesia were used to induce anesthesia and maintain anesthesia.The depth of anesthesia was satisfactory,and no mice died during anesthesia.(2)tail vein injection:the left tail vein injection was selected during intravenous injection.In group A and group B,no tail vein injection was performed;in group C and group D,9 patients were successfully injected into the tail vein,and the success rate was 90%.In group E,10 patients were successfully injected into the tail vein,and the success rate was 100%.(3)Success of model C57BL/6 mice:Group A was not modeled;Group B successfully modeled 9 with a success rate of 90%;Group C successfully modeled 8 with a success rate of 89%;Group D succeeded 9 models were built,the success rate was 90%;the E group successfully built 10 models with a success rate of 100%.(4)Death of mice in each group:0 deaths in group A,mortality was 0%;1 in group B and C,mortality was 11%;0 in group D and 0o mortality It is 0%.(5)The thrombosis rate of mice in each group:The thrombosis rate of mice in group A was 0%,and the thrombosis rates in mice in groups B,C,D,and E were 89%,87%,67%,and 70%,respectively.(6)Thrombosis wet weight,length,wet weight/length value of each group of mice:Group A mice were not modeled and were not plugged.The wet weights of thrombus in group B,C,D and E were 14.81±3.15 mg,13.50±08.73 mg,6.70±1.85 mg and 3.10±1.13 mg,respectively.Compared with group C,the wet weight of thrombus in group D was not statistically significant(P>0.05),and the wet weight of thrombus in group E was significantly reduced(P<0.05).The thrombus lengths of group B,group C,group D,and group E were 5.20±0.87 mm,4.15±1.93 mm,2.40±0.43 mm,and 2.38±0.83 mm,respectively.Compared with the length of thrombus in group C,the length of thrombus in group E was significantly shorter.,statistically significant(P<0.05).The wet weight/length of group B,group C,group D and group E were 2.85±0.41,3.01±0.63,2.91±1.25,1.35±0.56,respectively.Compared with group C wet weight/length,group E wet weight/length was obvious.Decrease,statistically significant(P<0.05).(7)The expression of Mmp-12 and Mmp-13 in the inferior vena cava of each group:Compared with the C group,the expression of Mmp-12-mRNA and Mmp-13-mRNA in group D and group E decreased,which was statistically significant(P<0.05).[Conclusion]1.KLF15 can regulate the expression of MMP-12 and MMP-13 on HUVECs.2.Mmp-13 can promote DVT formation in mice.3.Klf15 can regulate deep vein thrombosis by regulating the expression of Mmp-13. |
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