| Cardiovascular disease is a life-threatening disease as aged people becoming more and more in China. Thrombotic disease including acute myocardial infarction, stroke and pulmonary embolism, results in 26 millions people death every year. 10 millions people suffered thrombotic disease each year, and one million people dead, 3 million suffers need to be cured by drugs. Thrombotic disease has high incidence, disability, and mortality. Thrombolytic drugs are effective to cure thrombotic disease, but it has some side-effect and disadvantage such as short of half life, reocclusion, bleeding tendency, expensive and so on. Therefore, to develop new and novel style thromblytic drug has impotent significance.Plasminogen activator inhibitor 1 (PAI-1) is the main composition and it is one of the main regulators in the fibrinolytic system. PAI-1 inhibits tissue type plasminogen activator (t-PA) and urokinase type plasminogen activator (u-PA) to regulate the balance between fibrinolytic system and thrombotic system. Recent clinic and epidemiology survey demonstrated that over-expression of PAI-1 is an independent risk factor in the pathogenesis of coronary artery thrombosis and recurrent myocardial infarction, therefore, PAI-1 might be a new therapeutic potential target to prevent and cure cardiovascular disease. A new drug based on inhibition of PAI-1 expressions might be developed to treat thrombolytic disease. The application of antisense oligonucleotides (ASODNs) provide a new technology to treat thrombotic disease. Antisense technology involves in ASODNs, ribozymes, and small interfering RNA (siRNA). The principle for most application is to hybridize to their complementary RNA by Watson-Crick base pairing and inhibit target gene expression by a passive or active mechanism. The technology provides a novel approach in genomic function research and the screening of target of gene drugs.RNA is an ideal target to develop gene drugs. The screening of effective and specific sequence is the basis to devolop gene drugs. In this article, ASODNs against PAI-1 mRNA was screened by using full length gene targeting technology including screen in random oligouncleotide library, hybridization and RNase H-mediated cleavage on same microarray in situ. High potential activity siRNA sequences were designed and synthesized to investigate the thromblytic mechanism. The research has theoretical and practical significance to find and develop new gene thromblytic drugs.There are two parts in this article, one part is screening of ASODNs against PAI-1 by using full length gene targeting technology, the other part is screening of siRNA against PAI-1 and validation by cell biology.1. Screening of ASODNs against PAI-1 by using full length gene targeting technology and validation by cell biologyThe full length sequence of PAI-1 was cloned by RT-PCR, and its mRNA was transcripted in vitro and hybridized to random oligouncleotides library. A series of ASODNs complementary to the accessible sites located at full length region were screened and analysized by biology informatics. Then mRNA of PAI-1 was hybridized to ASODNs microarray, 15 of 21 ASODNs gave significant heteroduplex yield. 15 ASODNs were synthesized and validated by cell biology. ASODNs were transfected to human umbilical vein endothelial cells (HUVECs) at the concentration of 1.0umol/l in vitro, the results showed that 14 of 15 ASODNs significantly inhibited PAI-1 antigen after transfection 48 h, and the positive rate of was 93 %. 5 ASODNs (4# , 5#, 7# , 8# ,10#) which significantly inhibited PAI-1 antigen were transfected to HUVECs at the concentration of 0.1, 0.25, 0.5, 1.0 u mol/1 in vitro, the results showed these ASODNs significantly inhibited transcription, expression and activity of PAI-1.Screening of ASODNs against PAI-1 by using full length gene targeting technology could find effective and specific sequence, which reduced the probability of missing the accessible sites located at full length region and realized high-throught screening to find target ASODNs. ASODNs against PAI-1 mRNA could inhibit antigen expression and activity of PAI-1 by inhibiting protein translation from mRNA, the mechanism of ASODNs was different from tranditional thromblytic drugs. ASODNs might be developed to be a new therapeutic approach to thromblytic diseases.2. Screening of siRNA against PAI-1 and validation by cell biology5 ASODNs were obtaided using full length gene targeting technology and validation by cell biology. 2 siRNA that based on ASODNs were designed at these the accessible sites located at full length region and connected with vector and transfected to HUVECs in vitro, in order to study the inhibitary rate of transcription, expression and activity of PAI-1. 2 siRNA significantly inhibited transcription, expression and activity of PAI-1 in a dose dependant manner at the concentration of 0.1,0.25,0.5, l.Oug/ml.A correlation was found in binding sites between ASODNs and siRNA, the effective and specific binding site of ASODNs was always the effective and specific binding site of siRNA. The sequences of siRNA was their complementary RNA by Watson-Crick base pairing, and designed in the effective and specific binding site of ASODNs of PAI-1 at full length region. siRNA significantly inhibited transcription, expression and activity of PAI-1, the mechanism involves in gene silent of PAI-1 mRNA. The effect of siRNA has the advantage of novel, specific, safe, long-time and endogenesis.5 ASODNs were obtaided by full length gene targeting technology, these ASODNs significantly inhibited transcription, expression and activity of PA1-1. ASODNs might be developed to a new-drug different from tranditional drugs to cure thromblytic diseases. 2 siRNA that based on ASODNs were designed at these the accessible sites located at full length region, siRNA significantly inhibited transcription, expression and activity of PAI-1 in a dose dependant manner. siRNA screening out by using full length gene targeting technology has unique advantages, and might be developed to high effective and specific gene drug. Our studies provide theoretical and experiment basis for investigateing the mechanisms of thrombotic disease and screening of novel style thromblytic gene drugs.
|
PDF Full Text Request