An Experimental Study Of Prophylactic And Therapeutic Effects Of Oxymatrine On Rat Corpus Cavernosum Fibrosis Induced By HTGF-β1 Gene

PartⅠThe Construction of Eukaryotic Vector Carrying Human Transforming Growth Factorβ1 (hTGF-β1) GeneObjectives: To establish an efficient and reliable method of constructing, purifying and propagating the recombinant eukaryotic vectors pcDNA3.1(+)-hTGF-β1. Through the study of construction of replication-deficient recombinant vector coding for hTGF-β1 gene, we could transfect it into corpus cavernosum smooth cells and let it last to express hTGF-β1 in these cells.Methods: Recombining DNA and gene clone technique were applied to construct recombinant plasmid pcDNA3.1(+)-hTGF-β1 which contains human transforming growth factorβ1 gene. The recombinant plasmid pcDNA3.1(+)-hTGF-β1 were extracted and then detected elementarily the positive cloning gene via single enzyme and double enzyme digestion. We used the method of PCR to expand hTGF-β1 target gene fragments for further detection, and then to be sequencing.Results: Comparing the sequencing results of DNA fragment of hTGF-β1 gene with standard sequence in gene bank, homology is 99.82% and therefore it means the construction of recombinant plasmid get success.Conclusions: The recombinant eukaryotic vectors pcDNA3.1(+)-hTGF-β1 was successfully constructed.PartⅡThe Expression of Eukaryotic Vector carrying hTGF-β1 Gene in Corpus Cavernosum Smooth CellsObjective: To study the expression of the eukaryotic vector containing hTGF-β1, which were transfected into corpus cavernosum smooth cells.Methods: Plasmid pcDNA3.1(+) containing human transforming growth factorβ1 gene were constructed and named pcDNA3.1(+)-hTGF-β1, then were transfected into the primary cultured CCSMCs under the mediation of LipofectamineTM 2000. Positive clones were selected by G418; the expression was verified by using RT-PCR, ELISA, Western blot and immunocytochemical stain. And the proliferation of the transfected CCSMCs was evaluated by MTT, FCM. The apoptosis of cells was detected by FCM.Results: RT-PCR, ELISA, Western blot and immunohistochemical staining of post-transgenic CCSMCs were hTGF-β1 positive. And moreover the expression of transgenic hTGF-β1 mRNA could continued to 4 weeks after the transfection in the Group pcDNA3.1(+)-hTGF-β1. MTT colorimetric method showed that the transfected hTGF-β1 had a positive effect on the proliferation of CCSMCs. FCM did not find the apoptosis of the cells.Conclusions: Transfection of pcDNA3.1(+)-hTGF-β1 can provide persistent expression in CCSMCs. The hTGF-β1 had a positive effect on the proliferation of CCSMCs.PartⅢThe Establishment of Penile Fibrosis Model in a Rat using Eukaryotic Vector pcDNA3.1(+)-hTGF-β1 expressing Human Transforming Growth Factor-β1Objectives: Transforming growth factor-β1 (TGF-β1) has been suggested to have an important role in corpus cavernous fibrosis and result erectile dysfunction. For further elucidation of TGF-β1 signaling in association with cavernous fibrosis, we developed a rat model of corpus cavernous fibrosis using ukaryotic vector pcDNA3.1(+)-hTGF-β1 which could express hTGF-β1.Methods: The mixture of pcDNA3.1(+)-hTGF-β1 and LipofectamineTM2000 were injected into the male Sprague-Dawley rats intracavernously. The models and these mechanisms of fibrosis were verified by using picrosirius polarization method, RT-PCR, and immunocytochemical stain. Intracavernous pressure responses evaluated the erectile function.Results: After being stained by HE and the picricacid-siriusred staining, the slice was observed under polarized microscope at 7, 14, 21, 28 days postinjection showed multiple fibrous scars, the feature and regularity of the collagen change in the rats injected with the pcDNA3.1(+)-hTGF-β1 (Group pcDNA3.1(+)-hTGF-β1) and the recombinant human TGF-β1 protein injected rats (Group hTGF-β1). Whereas no histological evidence of cavernous fibrosis was found in the control rats (Group pcDNA3.1(+)). RT-PCR showed the expression of hTGF-β1, CTGF, FN mRNA in Group pcDNA3.1(+)-hTGF-β1 and Group rhTGF-β1 were higher than the Group pcDNA3.1(+). And moreover the expression of transgenic hTGF-β1 mRNA could continued to 4 weeks after injection in the Group pcDNA3.1(+)-hTGF-β1. Immunohistochemical staining also revealed a higher expression of hTGF-β1 in Group pcDNA3.1(+)-hTGF-β1 than in Group rhTGF-β1 and Group pcDNA3.1(+). Intracavernous pressure responses induced by papaverine injection in rats revealed that the maximal intracavernous pressure was significantly lower in Group pcDNA3.1(+)-hTGF-β1 than in Groups rhTGF-β1 or Group pcDNA3.1(+).Conclusions: The plasmid pcDNA3.1(+)-hTGF-β1 sufficiently induced relatively longlasting cavernous fibrosis. This novel animal model may contribute to the future investigations of the pathogenesis of penile fibrosis associated with TGF-β1 signaling and the development of new therapeutics targeting this pathway. PartⅣThe Experimental Study of Prophylactic and Therapeutic Effects of Oxymatrine on Rat Corpus Cavernosum Fibrosis induced by hTGF-β1 gene Objectives: To investigate the prophylactic and therapeutic effect of oxymatrine (OM) on experimental corpus cavernosum fibrosis and to revealed the mechanism. Methods: By establishing the models of corpus cavernosum fibrosis induced by hTGF-β1 gene in rats, we observed the effect of oxymatrine on the expression of hTGF-β1, CTGF, FN mRNA, tissue collagen fibers, microscopic changes, tissue pathology and the erectile function. Results: There was a decline expression of TGF-β1, CTGF, FN mRNA and collagen fibers in oxymatrine group, compared to those of the control group. Erectile function was higher in OM intervention group than in mode group, while the progress of those in OM group was lower than that in Dexamethasone group. Microscopy showed that OM groups had less fibrosis accumulation than model group. Conclusions: Our results suggest OM has prophylactic and therapeutic effects in rat corpus cavernosum fibrosis induced by TGF-β1 gene, probably by protecting to inhibit the expression of TGF-β1 and its downstream factors, then inhibits the generation of the collagen fibers in the corpus cavernosum of the rat.