The Study Of Transcription Factor KLF2 And KLF4 For Predictive Diagnosis Deep Vein Thrombosis

ObjectiveTo study the expression of transcription factors KLF2 and KLF4 mRNA in the prethrombotic,thrombosis,non-thrombosis status of rat traumatic deep vein thrombosis(TDVT) model blood and vascular tissues,and feasibility as new predictive molecular markers to diagnosis deep vein thrombosis(DVT),analysis the role of KLF2 and KLF4 in the thrombosis,lay a foundation for further study of DVT molecular mechanism and regulatory network.Methods1.Summarize and analyze Rat femoral veins microarray data①The establishment of rat TDVT model,animal grouping,experimental procedur,femoral veins Affymetrix 230A microarray detection has been discussed in Dr.Wang Bing's thesis "Establishment of a rat acute traumatic deep vein thrombosis model and femoral vein wall gene expression study ".②Summarized and analyzed the microarray data,applied fold change method to scan and analysis differential expression genes involved in DVT.③Analyzed the expression tendency of KLF2 and KLF4 in rat vascular tissue of DVT.④Queried both rat and human gene sequences of KLF2 and KLF4 in the NCBI GENE website,than used BLAST to determine the homology.2.Rat TDVT model blood Real Time-PCR detection①According to different observation phases and thrombosis observation results, 100 TDVT model SD rats were divided into 4 groups:the control group(group A), prethrombotic group(group B),thrombosis crest-time group(group C), non-thrombosis group(group D).②In the corresponding time,2.0ml blood samples were hemospasia from heart of each model,and cut bilateral femoral veins.③Used TRIzoI method to extracte total RNAs of blood,the qualities of RNA samples were evaluated by 3%agarose gel electrophoresis and spectrophotometer detection.④Applied Oligo 6.69 software was to design KLF2 and KLF4 PCR primers,with GAPDH as an inner-reference.Each group blood cell RNA was reversed transcription into cDNA,and then PCR reaction was performed(force--denaturalization--degeneration --annealing--extension--end-extension).The PCR production was analyzed by fluorescent quantitative method.Identified KLF2 and KLF4 specific amplification products by melting curve and detected the amount of products by amplification curve.⑤Data processing method of Real Time PCR:2^{-ΔΔCT} method, the 2^{-ΔΔCT} result is a ratio experimental group(group B,C,D) versus control group (group A) of KLF2 and KLF4.Then,analysis both of them expression tendency in rat blood of DVT.⑥Applied SPSS 11.5 software for statistical analysis,P＜0.05 as statistically significant.Result1.In the Affymetrix gene chip data of rat TDVT model femoral vein,KLF2 and KLF4 had a same down-regulated expression trend from post-traumatic instant (group B) to thrombosis crest-time group(group D)(KLF2-BvsA:-0.104,CvsA: -0.715,DvsA:-1.565;KLF4-BvsA:-0.054,CvsA:-0.834,DvsA:-1.276).Group D compared with the control group(group A) were significant differences in down-regulated expression.Non-thrombosis group(group G) was up-regulated slightly(KLF2-GvsA:0.135;KLF4-GvsA:0.245).2.Both KLF2 and KLF4 in rat and human genes BLAST homology results were 90.4%and 94.2%.3.The DVT rate of rat TDVT model after operated 25h was 63.39%(42/66),which was no significant difference(P=0.405＞0.05) with the rate of microarray detection rat model(68.11%,126/185);and the non- thrombosis rate after 72h was 63.39% (42/66),which was no significant difference(P=0.282＞0.05) with the rate of microarray detection rat model(19.46%,36/185).4.Rat TDVT model blood Real-Time PCR and femoral vein microarray detection results of KLF2 and KLF4 presented similar trends(Z=-1.363,P=0.173＞0.05). Both prethrombotic group(group B) and thrombosis crest-time group(group C) compared with the control group(group A) were significant differences in down-regulated expression(KLF2-BvsA:-1.510,CvsA:-2.889;KLF4-BvsA: -1.262,CvsA:-2.041),while non-thrombosis group(group D) was up-regulated (KLF2-DvsA:0.796;KLF4-DvsA:0.612).Conclusion1.KLF2 and KLF4 presented similar trends in rat TDVT model blood and vascular tissue,and can be used as candidate molecular markers to predict diagnosis DVT.2.KFL2 and KLF4 play an important role of transcriptional regulation in DVT regulatory network.They were key factors of vascular endothelial cells steady-state regulation and signal transduction pathways,by inhibiting vascular inflammation,promote the expression of anticoagulant factors and other roles to inhibit thrombosis.3.KFL2 and KLF4 may be as keys "molecular switch" that regulates important aspects of vascular function and thrombosis disease,and expected to become new predictive diagnosis molecular markers and prevention and treatment drug targets of DVT in clinical.