Dissertation
Dissertation > Medicine, health > Basic Medical > Human biochemistry, molecular biology

The Exploration of the Treatment to ED by Knocking Down the Expression of PDE5 in Smooth Muscle Cells of Human Corpus Cavernosum Through siRNA

Author ZhanYing
Tutor LiuJiHong
School Huazhong University of Science and Technology
Course Surgery
Keywords PDE5A3 PDE5 siRNA RNAi Adenovirus Corpus cavernosum smooth muscle cells Rat
CLC R346
Type PhD thesis
Year 2007
Downloads 93
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Objective: To construct specific siRNA, plasmids and viral vectors using the inhibition of human and rat corpus cavernosum smooth muscle cells PDE5 gene expression studies RNAi gene therapy in the ED's role. Method: ① According select two people PDE5A3 mRNA target sequence to synthesize complementary oligonucleotide strands, respectively, after the band enhanced green fluorescent protein (EGFP) gene marker pEGFP6-1, pGenesil-1 plasmid vector and transformed into bacteria, expanding increased after purification to obtain the desired plasmid. Transfected human corpus cavernosum smooth muscle cells were observed under a fluorescence microscope transfection results, transfection efficiency was measured by flow cytometry. ② By screening the human PDE5A3 gene inhibition of three siRNA expression cassettes with enhanced green fluorescent protein (EGFP) expression cassette was cloned into both eukaryotic expression vector pGenesil-1 in pShuttle2 then subcloned into the shuttle plasmid. By homing endonuclease I-CeuI/PI-SceI subcloned order to express the purpose of adenovirus backbone vector pAdeno-X, the resulting carrying three siRNA expression cassette and EGFP recombinant plasmid expression cassette. Confirmed by PCR and restriction enzyme linearized with PacI and transfected cells HEK293A packaging adenovirus particles. EGFP expression was observed gene was amplified by PCR and other methods to be identified. Half of tissue culture infective dose (TCID50) virus titer determination. ③ transfected in vitro primary cultured human corpus cavernosum smooth muscle cells 48h and 72h, using RT-PCR and Western blot assay cavernosum smooth muscle cells PDE5A3 gene expression levels. ④ primary cultured rat corpus cavernosum smooth muscle cells, PCR cloning its PDE5 gene, the nucleotide sequence according to its target sequence selection, design and synthesis of 6 siRNA fragments and prepare six siRNA expression plasmid. The PDE5 gene expression cassette was subcloned into pIRES2 labeled with EGFP plasmid vector to construct double gene co-expression vector PIRES2-EGFP-PDE5. The vector with respectively 6 siRNA expression plasmids were co-transfected HEK293A cells for 48h by fluorescence microscopy and radiography, RT-PCR and Western blot analysis PDE5 mRNA and protein expression. Will be screened on rat PDE5 inhibition of gene expression cassettes with two siRNA enhanced green fluorescent protein (EGFP) expression cassette was cloned into both eukaryotic expression vector pGenesil-1 in pShuttle2 then subcloned into the shuttle plasmid. Was constructed to carry two different siRNA expression cassette plasmid and adenovirus titer was measured in animal models for further research foundation. Results: The enzyme electrophoresis and DNA sequencing confirmed and siRNA eukaryotic expression vector was constructed successfully. Corpus cavernosum smooth muscle cells transfected after 48h, visible under a fluorescence microscope bright green fluorescence, the average transfection efficiency was 17.95%. Construction of recombinant adenovirus vector rAd5-siRNA-PDE5A3, sequencing showed that the target gene sequence and in the correct orientation, restriction enzyme digestion and PCR fluorescence observation proved successfully constructed, the virus titer of 8.0 × 108pfu/ml. After all HEK293A adenovirus infected cells were GFP expression. Amplification and purification of recombinant rAd5-siRNA-PDE5A3 and control virus Ad5-EGFP, transfection of human corpus cavernosum smooth muscle cell efficiency up to 95% or more, PDE5A3 gene expression at the mRNA level was suppressed 48h (71.7% ± 2.30)% , 72h was (70.8 ± 3.61)%, 48h was inhibited at the protein level (78.3 ± 3.33)%, 72h was (77.5 ± 2.67)% (P lt; 0.01). 6 rat PDE5 specific siRNA recombinant plasmid and rat PIRES2-EGFP-PDE5 double gene co-expression vector was confirmed by restriction analysis and sequencing successful construction of specific siRNA expression plasmid transfected group was significantly weaker than the control EGFP fluorescence intensity groups. RT-PCR and Western blot showed that the recombinant plasmid Pgenesil-2-siRNA4 PDE5 expression in rat most significant, mRNA and protein expression inhibition rate was (68.59 ± 3.23)% and (71.25 ± 2.91)%. Followed Pgenesil-2-siRNA6, on PDE5 mRNA and protein expression inhibition rate was (55.02 ± 3.44)% and (65.09 ± 3.05)%. No. 4 and No. 6 will continue to build a viral vector plasmid and sequencing showed that the target gene sequence and in the correct orientation, restriction enzyme digestion, fluorescence observation and PCR results show rAd-siRNA-rPDE5 adenovirus vector was constructed successfully, the virus titer of 8.0 × 108pfu/ml. After all HEK293A adenovirus infected cells were GFP expression. Conclusion: The successful construction of two gene-specific inhibition against PDE5A3 siRNA eukaryotic expression vector, and can be effectively expressed in the target cells. With three people PDE5A3 gene siRNA fragments adenovirus vector was successfully constructed, expressed in eukaryotic cells by shRNA could significantly inhibit human corpus cavernosum smooth muscle cells PDE5A3 gene expression; successfully constructed and rat-specific siRNA expression plasmid PIRES2-EGFP-PDE5 double gene co-expression vector and co-transfected HEK293A cells. RNAi could significantly inhibit PDE5 rat corpus cavernosum smooth muscle cell gene expression, rapid screening of effective siRNA sequences and successfully construct containing two rat recombinant PDE5 gene siRNA fragments for further study of gene therapy for ED provided the experimental basis .

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