| Penile erection depends on the integration of vascular, endocrine and neurological mechanisms. Neurally mediated vascular dilatation and relaxation of penile smooth muscle leads to increased blood flow and erection of the cavemosal tissue. Vasoactive intestinal polypeptide (VIP) is a potent vasodilator and smooth muscle relaxant, and fulfils several of the criteria for a neurotransmitter mediating penile erection. Erectile dysfunction is a common comorbidity in patients with diabetes. As many as 75% of diabetic men will be confronted at some time in their lives with a consistent or recurrent inability to achieve and maintain an erection adequate for sexual performance, typically at an earlier age than their counterparts with normal glycemic control. In one cohort study, erectile dysfunction affected >47% of men with type 1 diabetes aged ≥43 years, compared with 1.1% of those aged 21-30 years. According to one estimate, >50% of men will develop erectile dysfunction within 10 years of diabetes onset. Erectile dysfunction and diabetes each affect >150 million people worldwide, and this value is projected to double by the year 2025. The mechanisms responsible for diabetes-induced erectile dysfunction are controversial. VIP has been no agreement about its effects ondiabetes-induced erectile dysfunction since some studies have found decreased VIP and diminished VIP never fibres. A variety of additional mechanisms including central neuropathy and decreased levels of circulating androgens have also been proposed to explain the erectile disorders found in diabetic rats. The present study was devised to determine whether transfection of VIP cDNAto the corpus cavemosum of the penis may result in correcting erectile dysfunction without affecting other important organs in diabetic rats. As VIP is one of the main neurotransmitters for penile erection, the introduction of it may result in improving erectile function.Part OneConstruction of Recombinant Plasmid Vector Coding VasoactiveIntestinal Polypeptide GeneObjectives: To construct of recombinant plasmid vector that codes vasoactive intestinal polypeptide (VIP) gene.Materials and Methods:1. Primers:VIPF1: 5'-CCAAGCTTATGAGTTCCTGGCGATCCTG-3'VIPR1: 5'-TTGGATCCTCATCATTTCTCTAGCTCTTC-3' VIPF2: 5'-CCTGGCGATCCTGACACTCTTC-3' VIPR2: 5'-CTCTAGCTCTTCAAGGAAGTCT-3'2. RT-PCR: RNA extraction; For RT-PCR, total RNA was reversed transcribed, total RT reaction was used as template cDNA for PCR. PCR was performed with VIP mRNA specific primers for 30 cycles with cycletimes of 2 minutes at 94℃ (denaturation), 54℃ 1 minutes (annealing), 72 ℃ 45 seconds (extension), the last cycle extension for 10 minutes. 3. The VIP cDNA (-500 nucleotides; i.e., 0.5kb) was inserted into the pcDNA3.1 vector, where expression is driven off of the cytomegalovirus promoter. Results: PCR and double restriction enzyme digestion showed that therecombinant vector, pcDNA3.1/VIP, was constructed correctly. Conclusions: The pcDNA3.1/VIP containing rat VIP cDNA sequence has been constructed successfully, thus providing an importont and convenient tool to study on gene transfer of neurotransmitter VIP to penis in the diabetic rats.Part TwoThe Foundation of Diabetic Animal ModelObjectives: To found the diabetic animal model for the study of VIP gene transfer to diabetic rat penis.Materials and Methods:1. This study was conducted on 120 male Sprague Dawley rats, 10 weeks old, body weight 280±23g. Rats were maintained in alternating cycles of darkness (6.00 p.m. to 6.00 a.m.) and light (6.00 a.m. to 6.00 p.m.). Food and water were freely available;2. Streptozotocin, N-[Methylnitrosocarbamoyl]-D-glucosamine, STZ;3. Protamine zinc insulin;4. SD rats were divided into two groups: Experimental group (n=108): treated with a single intraperitoneal injection 3 ml of STZ (60mg/kg, diluted in 0.1 M, pH 4.5 sodium citrate buffer); Control group (n=12): treated with a single intraperitoneal injection 3 ml of sodium citrate buffer (0.1 M, pH 4.5);5. Bloo...
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