| Chapter 1 The design of specific triplex-forming oligonucleotides to rat uPA geneSection 1 Bioinformatics analysis and prediction of promoter of rat uPA geneUrokinase-type plasminogen activator (uPA) is well related with the male reproduction, however, the mechanism has not been completely elucidated. In this chapter, a system bioinformatics evaluation of rat uPA gene and protein was performed. The rat uPA cDNA closely resembles that of the other uPAs, sharing an average of 86, 76, 76, and 70% sequence homology with the mouse, human, baboon, and pig uPA cDNAs, respectively; nucleotide homology is highest within the coding sequences (91, 88, 83, and 76%), although the 3' untranslated regions are more divergent. The uPA cDNA contains a single large open reading frame encoding a 432-amino acid protein with a predicted molecular weight of 48,000, in keeping with PA gel zymography data. Amino acid sequence homology with the other uPA proteins is high (88,71,70, and 69%, as above) and considerably higher if conservative amino acid substitutions are ignored (98, 93, 93, and 84%). Unlike the human and pig uPA proteins, rat uPA has no potential sites for glycosylation but retains the conservation of sequence in the epidermal growth factor (EGF), Kringle, and serine protease domains exhibited by all the uPAs. Application of TFOs requires the presence of long possibly uninterrupted polypurine sequences in the target DNA to ensure optimal stability and sequence specificity. Such purine-rich tracts are frequently found in gene promoter regions and frequently overlap transcription factors binding sites. Therefore, the purine-rich elements may be relevant for control of gene expression and can be relatively easy targets. The bioinformatics analyisis of rat uPA showed that a nonclassical TATA box located 27 bp upstream the transcription initiation site and adjacent GC boxes, potential Sp1 and AP-1 binding sites could been found, indicating a core promoter in this region. In addition, within the predicted core promoter region, a 25 mer polypurine / polypyrimidine rich sequence was located upstream 81 bp upstream of transcription initiation site and 47 bp upstream of TATA box as potential TFOs targeting sequence.Section 2 Evaluation of affinity of TFOs by electrophoretic mobility shift assaysIn this section, the stable triplexes, formed by rat uPA specific triplex-forming oligonucleotides (TFOs) under experimental conditions, were tested by means of electrophoretic mobility shift assays (EMSA). EMSA was performed using a 35-mer duplex containing the polypurine / polypyrimidine sequence of the rat uPA gene as a DNA targe. The target was prepared by annealing the [γ-32P] ATP end-labeled pyrimidine strand with a slight excess of the complementary purine strand and then mixed with an excessive amount of TFOs. The results indicated that although both uPA-PO and uPA-PS possessed the mobility of the 25-mer-target duplex, however, the latter was much less than the former in quantity and the Kd of uPA-PO was 10-7 M and uPA-PS was 10-6 M.Chapter 2 Primary culture and identification of Sertoli cellsPrimary culture is a useful tool to study the spermatogenesis process and the interactions between Sertoli-Sertoli cells and Sertoli-germ cells. In this part, primary culture system of Sertoli cell was established through sequential enzymatic treatment using 0. 25% trypsin and 0.1% collagenase and 0.1% hyaluronidase and thereafter the cell suspension was cultured in incubator under the condition of 32℃and 5% CO2 for 48 h, then Sertoli cells were treated by hypoosmotic Tris-Cl (pH 7.4)solution and cultured continuously. Sertoli cells were identified through several methods as well. All together, with 95% purity of Sertoli cells, Sertoli cells culture system we established was competent to the following experiments.Chapter 3 TFOs Downregulates uPA Transcription TFOs Downregulates uPA Transcription in rat Sertoli cells in vitroSection 1 The design of the primer and establishment of the method on SYBR Green I rea1-time quantitative RT-PCR for detecting the expression of urokinase-type plasminogen activator mRNA in ratsIn order to design a pair of primers and to establish the method of SYBR Green I based real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) for detecting the expression of Rattus norvegicus urokinase-type plasminogen activator (uPA) mRNA.The mRNA sequence was drawn from gene bank and related bioinformation was collected and analysised thoroughly. Then uPA mRNA was amplified by using RT-PCR and the amplicon was purified and diluted by 10 times in order to establish standard curves. The components of PCR system and conditions were optimized according to the analysis of melting curves,amplify efficiencies, correlation coefficients and the slope of standard curves.On the basis of all the data, the 21bp primers were designed with software Oligo 6.22 which contained 52.4% GC content. The most stable 3'-dimer and hairpin stemsof upper and lower primer were -1.5, -0.40 kcal/moland -3.5, -0.90 kcal/mol respectively. Upper-Lower 3'-duplexes was -3.1 kcal/mol. Upper-lower primers possessed the property that 5' and middleΔG were higher than 3'ΔG and their priming efficiency was 455 and 403. Employing the primer, the optimized is: a fixed amount of 2μl RT products (20ng) and 200nmol/L upper-lower primers were co-amplified at a final concentration of 1×PCR buffer, 200μmol/L dNTPs, 3mmol/L MgCl2 0.05unit/μl of thermos aquaticus DNA polymerse and 0.2×SYBR Green I in a total volume of 25μl. The PCR mixture was amplified for 38 cycles with Mx3000pTM PCR thermal cycler. The amplifyication profile was involved in predenaturation at 95℃for 5min, denaturation at 95℃for 30s, primer annealing at 58℃for 20s, and extension at 72℃for 25s. The standard curve with a efficiency range of 101.0～108.7% (a slop of -3.299～-3.13 accordingly) and its correlation coefficient was 0.996～0.999. The linear range of PCR could been obtained within 6 logs. Additionally, the stability of the method was examined.The intra and inter run variability were 0.05%,0.02%,1.7% and 1.78%,2.29%,3.54%, respectively. All together, the primers were proved to be appropriated and could specifically and efficiently detect the target sequence and the optimal real-time PCR with SYBR Green I is fit to detect the expression of uPA mRNA with good sensitivity, specifity, repeatablity and linear range.Section 2 TFOs Downregulates endogenerous uPA TranscriptionUrokinase-type plasminogen activator (uPA), expressed in Sertoli cells in the testis, is closely related with tight junctions of blood testis barrier (BTB); and it has been considered as a potential contraceptive target. In the present study, the antigene effects of triplex-forming oligodeoxynucleotides (TFOs) targeting uPA in rat Sertoli cells were investigated in vitro. Although tPA, another form of plasminogen activators (PAs), partially compensated the lose of PAs activities, uPA mRNA and protein were significantly reduced as demonstrated by real-time reverse transcription PCR and a chromogenic assay, after the treatment of Sertoli cells with uPA specific TFOs at a concentration of 330 nM. The capacity of TFOs resistance to nuclease degradation was enhanced by the phosphorothioated on the backbone of the oligonucleotides. Our results indicated that the TFOs can down-regulate uPA expression and uPA might be an alternative contraceptive target.
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