The Study Of The Expression Of TF Gene In Endothelial Cells Regulated By The Blood Flow Shear Stress And TFO

Cerebral atherosclerotic thrombosis is one of the most common cerebrovascular disorders. With the development of living level, the incidence of the cerebral atherosclerotic thrombosis presents higher than before. The complication especially cerebral infarction resulted from cerebral atherosclerotic thrombosis is hard to be cured and influences people s life and work seriously. By far, the effect of various treatments aimed to the disorder are not efficient. Only about 20%~~30% inhibitory efficiency of drugs which prevent platelet from congregating on cerebral thrombosis is confirmed. Therefore, it is necessary to search a new treatment to prevent cerebral atherosclerotic thrombosis efficiently.Tissue factor (TF) is an important factor which regulates the process of hemostasia and thrombosis. Under the physiological conditions, the endothelial cells express TF gene very little to maintain the normal function of the blood circulation. While under the pathological conditions, the endothelial cells with functional disorder may express TF gene abnormally, which brings the endothelial cells from the anticoagulant state into the procoagulant state. More recently the role of TF gene has been paid more attention to the vascular thrombosis diseases such as atherosclerosis (AS).It is known that the cerebral atherosclerotic thrombosis usually occurs in the narrow, curved and divergent areas of brain blood vessel, or in valley of atherosclerotic plaque, which suggests that the hemodynamic factors especially shear stress play an important role in the cerebral atherosclerotic thrombosis and distribution. Further studies indicate shear stress regulates TF gene expression via the endothelial cells. It has been indicated that the remarkable changes of local shear stress induced the expression of TF gene, which is the key role in the start and development of the local intravascular thrombosis. Therefore, down-regulating the expression of TF gene in the endothelial cells may become a primary approach in the prevention of the local intravascular thrombosis.Human TF gene is located on chromosome 1 (Ip21~lp22), whose span of cDNA sequence is 2.3Kb. TF promoter contains three overlapping Sp1/Egr-1 binding site andspans a region between -111 and +15bp (relative to the start site of transcription) which named shear stress responsive element, SSRE. They are specific cis-action regulatory elements for shear stress to induce the expression of TF gene. However, how the shear stress regulates the interaction of the transcriptions and SSRE is unclear. It is considered great significance to inhibit the activation of SSRE efficiently which suppresses the expression of TF gene induced by shear stress and prevents the local intravascular thrombosis. It is considered that inhibiting the activation of the SSRE in TF promoter would prevent the expression of TF gene induced by shear stress, and antigene technology provides an efficient way for it.Triple helix-forming oligonucleotide (TFO) is designed by the antigene technique. After modified, the TFO can penetrate the plasma membrane directly and bind to the duplex DNA via Hoogsteen hydrogen bonds rather than the mRNA which can inhibit the gene transcription more effectively. Previously we have designed and constructed TFOs according to the three overlapping Spl/Egr-1 sites in the TF promoter, and primarily selected 3 apTFOs (antiparallel-phosphorothioated TFO) by the electrophoretic mobility shift analysis (EMSA) which showed high affinity to the Spl/Egr-1 sites. The 3 apTFO, T14GTa-ps, T21GTa-ps and T15GTa-ps, are respectively according to the 1st, 2nd and 3rd site of the Spl/Egr-1 sites. However, it is required to further prove the effect of the 3 apTFOs. At the same time, how the shear stress regulates the interaction between the transcriptions (Spl and Egr-1) and SSRE is still unclear which required further study too.In the first part of the study, the 3 apTFOs' inhibitive effect on the expression of TF gene were evaluated by report gene and RT-PCR. In the second part it was to test how th...