| DVT(Deep Venous Thrombosis,DVT)is a cardiovascular disease with high mortality,complex treatment,multiple factors and multiple regulatory mechanisms.Its pathogenesis involves the endothelium of blood vessels,components in blood,as well as the fibrinolysis system.The expression changes of IL?Argl?P-Selectin?Hmoxl?TF?Mmps and many other genes in venous endothelium play an important role in the formation of DVT.Studies have shown that the Mmps family is involved in the degradation and remodeling of extracellular matrix in vascular walls,which is related to many physiological and pathological processes of vascular diseases.Therefore,the purpose of this study is to:1.Using Klf15 gene knockout homozygous C57BL/6 mice to further verify the effect of Klf15 on Mmp19 expression;2.To explore the effect of Mmp19 on deep venous thrombosis in mice.[Objective]1.To cultivate Klf15 gene knockout homozygous C57BL/6 mice and make DVT model by inferior vena cava stenosis to compare the differences of Mmp19 expression in deep vein thrombosis and vascular wall between Klf15 knockout mice and wild type mice,thus to further verify the effect of Klf15 on Mmp19 expression.2.Mmp19-siRNA was injected into caudal vein to interfere the expression of Mmp19 in mice,then DVT model was established in Klf15 gene knockout homozygous C57BL/6 mice,and the changes of Mmp19 expression in venous endothelial tissue of mice were detected to explore the effect of Mmp19 on the formation of DVT in mice.[Materials and Methods]The first part:To create Klf15 knockout homozygous C57BL/6 Mice by CRISPR/Cas-mediated genome engineering1.Strategy of genome engineeringKlf15 gene is located on chromosome 6 of C57BL/6 mice,and its starting codon ATG" is in Exon 2,and the termination codon "TGA" is in Exon 3.We selected Exon2 as the target to design gRNA,by means of high-throughput electric transfer of fertilized eggs to obtain Klf15 gene knockout heterozygous fertilized eggs,bred mice,genotyped baby mice by PCR,and confirmed by sequencing that Klf15 gene was knocked out.3.To produce homozygous Klf15 knockout mice by heterozygous Klf15 knockout mice1)Genetically engineered mice selected for sexual maturity(7-8 weeks)were mated with wild-type mice.a.If F0 is male,long-term cohabitation method of 1:3 can be adopted for pregnancy.b.If F0 is female,adopt 1:1 long-term cohabitation method for mating.If female rats still have no signs of pregnancy after 5 weeks,replace wild-type male rats.2)Identification of F1About 7 days old,the baby mice can be identified by the method of PCR or(and)sequencing for identification with cutten tail,and the baby mice can be numbered independently.The offspring are theoretically 50%engineered mice.3)Row F2 homozygous reproductionOne of F0 chromosomes is mutated,that is,it is completely consistent with the genotype of offspring obtained from wild-type mating.For F2 generation homozygous reproduction,only F1 males and females obtained from the same F0 are matched in cages3.Homozygous identification,Klf15 gene was obtained to knock out homozygous miceThe second part:Generation of ?C thrombus in Klf15 gene knockout homozygous C57BL/6 mice to investigate the effect of Mmp19 on formation of formation in thrombus.1.The experiment was divided into two parts:in the first part,the mice were divided into three groups(Blank group,DVT group and Klf15-KO group).Six wild type C57 mice and three homozygote mice were selected.Six wild type mice were randomly divided into two groups:Blank group(n=3)and DVT group(n=3).In addition,3 Klf15 gene knockout homozygous C57BL/6 mice obtained in experiment 1 formed Klf15-KO group(homozygote,nasty 3),mice were reared in SPF animal room,regardless of sex,weighing about 24-26 g.The model of inferior vena cava stenosis was made in DVT group and Klf15-KO group.24 hours after operation,thrombus and vascular endothelial tissue at the site of thrombus formation were dissected.Real-timePCR was used to detect the expression of Mmp19 in venous endothelium of mice in each group,and the thrombus formation rate,length,wet weight and other related indexes were measured.In the second part,12 Klf15 gene knockout homozygous C57BL/6 mice were randomly divided into 4 groups:Blank group(group A,n=3),DVT-KO group(group B,n=3),Mmp19-NC group(group C,n=3)and Mmp19-siRNA group(group D,n=3).In addition,3 C57 wild-type mice with the same body weight were randomly selected as DVT-WT group(group E,n=3).Three mice in Mmp19-siRNA group were injected with Mmp19-siRNA inhibitor into tail vein.Three mice in the Mmp19-NC group were injected with normal saline into the tail vein.24 hours after the tail vein injection,the DVT model was made by stenosis.The thrombus and the vascular endothelial tissue at the site of thrombus formation were also dissected after operation.Real-timePCR was used to detect the expression of Mmp19 in the vein endothelium of each group,and the thrombus formation rate,length,wet weight and other related indexes were measured.2.The data were processed and analyzed by SPSS 22.0 statistical software.[Result]Experiment 1:To create Klf15 knockout homozygous C57BL/6 Mice by CRISPR/Cas-mediated genome engineeringBy intercrossing heterozygous Klf15 knockout mice,we generated totally 16 Klf15 knockout homozygous mice for further experiment from 25th October 2018 to September 13,2019.Experiment 2:Generation of ?C thrombus in Klf15 gene knockout homozygous C57BL/6 mice to investigate the effect of Mmp19 on formation of formation in thrombus.The first part of the experimental results? Modeling and survival:there were no models in the Blank group,and 3 models were successfully made in the DVT group,with a success rate of 100%in the Klf15-KO group,with a success rate of 100%.The mortality rates of Group A,B and C were all 0%.?Thrombus formation rate:there were no models in Blank group;2 thrombus were composed of DVT,and the thrombus formation rate was 66.67%for both DVT and Klf15-KO group.? Wet weight,length,wet weight/length of thrombus in each group:the wet weight of vascular tissue without thrombus formation in Blank group was 3.07±0.77,vascular tissue.The wet weight of vascular tissue and thrombus in DVT group and Klfl5-KO group were 12.77±1.54mg and 30.35±1.65mg,respectively.Compared with the wet weight of thrombus in DVT group,the wet weight of thrombus in Klf15-KO group was significantly higher.The tissue length of Blank group,DVT group and Klf15-KO group was 2.48±0.47mm,4.71±0.12mm,4.40±0.19mm respectively.The wet weight/length ratio of Blank group,DVT group and Klf15-KO group was 1.21±0.07,2.70±0.26,6.95±0.65 respectively.Compared with DVT group,the wet weight/length ratio of Klf15-KO group was significantly higher.?Expression of Mmp19 in inferior vena cava of mice in each group:compared with the DVT group,the expression of Mmp19 in the Klf15-KO group was significantly higher than that in the Klf15-KO group(P<0.05).The second part of the experimental results? Caudal vein injection:the left tail vein injection was selected during intravenous injection.The successful caudal vein injection was 2 in group C and 3 in group D.?Modeling and survival:there were no models in group A;3 models were successfully made in groups B,D,E and 2 in group C.There were 0 deaths in groups A,B,C,D and E,and the mortality rate was 0%.? Thrombus formation rate:there were no DVT models in group A,2 in group B,C and E with the rates of 66.67%.There were 3 in group D with the rate of 100%? Wet weight,length,wet weight/length of thrombus in each group:the wet weight of vascular tissue without thrombus in group A was 0.34±0.04mg.The wet weight of vascular tissue and thrombus in group B,C,D and E were 3.77±0.54mg,3.35±0.48mg,1.63±0.11mg,1.87±0.44mg respectively.Compared with the wet weight of thrombus in group C,the wet weight of thrombus in group D was significantly lower(P<0.05).The tissue length of group B,C,D and E were 4.64±0.05mm,4.48±0.11 mm,4.45±0.30mm,4.63±0.10mm respectively.The wet weight/length of Blank,B,C,D and E groups were 8.12±1.09,7.50±1.24,3.74±0.47,4.06±1.04 respectively.Compared with group C,the wet weight/length ratio of group D was significantly lower(P<0.05).? Expression of Mmp19 in inferior vena cava of mice in each group:compared with group C,the expression of Mmp19 in group D was significantly lower(P<0.05)[Conclusion]1.Klf15 leads to repression of the Mmp19 expression in mice.2.Mmp19 in the vein endothelium of mice promotes the formation of DVT. |
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