Gene Transfer Of Calcitonin Gene-related Peptide To Penile Corpora Cavernosa And The Effect On Erectile Function In Diabetic Rats

Part I Construction of eukaryotic expression vector coding rat calcitonin gene-related peptide(pcDNA3.1/rCGRP)ObjectiveIt is to construct the recombinant plasmid vector that codes calcitonin gene-related peptide(CGRP) of rat(pcDNA3.1(+)/rCGRP). The vector is to be applied to the purpose for gene therapy of diabetic erectile dysfunction.MethodsA pair of primers with restriction enzyme sites was designed according the rCGRP gene order in the Genbank. The total RNA was extracted from the brain tissue of rat with Trizol reagent, and the rCGRPcDNA was amplified by RT-PCR. The PCR products and pcDNA3.1(+) were digested with restriction enzymes, purified and ligated; Recombinant colonies were harvested and identified by PCR, restriction enzyme digestion and DNA sequencing.Results(1)analysis of total RNA The OD260/OD280 ratio of the extracted total RNA was 1.8497. Two distinct bands of 28S and 18S were evident with the electrophoresis of total RNA under 1.5% agarose gel.(2)electrophoretic analysis of RT-PCR product Under the 1% agarose gel electrophoresis, the amplified products of RT-PCR present a specific band that was coincided with the size of rCGRPcDNA.(3)Identification of the recombinant colony â‘ identification with PCR Under the agarose gel electrophoresis, the amplified products of PCR present a specific band that coincided with the size of rCGRPcDNA. â‘¡identification withrestricted digestion The extracted plasmids were digested by two restriction enzymes, and the digested products presented two specific bands thosecoincided with the sizes of pcDNA3.1 and rCGRPcDNA respectively; â‘¢DNAsequencing The recombinant plasmid identified by the former steps was sent out for direct DNA sequencing. The acquired sequence was analyzed by BLAST and demonstrated to be completely identical with the coding region sequence of rCGRP.Conclusion(1)There existes gene expression of CGRP in the brain tissue of rat. (2)The pcDNA3.1(+)/rCGRP is successfully constructed with the techniques of RT-PCR and gene recombination.Part II Modeling diabetic rat and testing its erectile functionObjectiveIt is to model diabetic rats with erectile dysfunction. The rats are supplied to the following sdudy on gene therapy of diabetic erectile dysfunction.Methods150 Sprague-Dawley male rats, weighing 200-250g, were randomly divided into experimental group(n=138) and control group(n=12). The rats of experimental group were intraperitonealiy injected with streptozotocin(STZ) at 60mg/kg, and the control group rats were injected with volume-mached 0.1M natrium citricum(Ph4.5). After 3d of the injection, the blood was collected from tail veins and blood glucose levels were determined. From then on, the blood glucose levels were determined for every two weeks. DM was diagnosed according to the glucose levels and symptoms of the rats. After 10wk of the injection, 12 rats were randomly selected from the successfully-modeled DM rats and their rCGRPmRNA and CGRP expressions and intracavemous pressures(ICP) were determined and compared with those of control group.Results(1)General conditions In the experimental group, 3 rats died after 5, 7, and8wk respectively because of severe DM. There were 4 rats whose glucose levels were under the creterion at 8wk and 10wk, and the rats were regarded to be resisted to STZ and excluded from the successful DM models. At last, 131 of the 138 rats(regarded as DM group) were successfully modeded and the achievement ratio was 94.9%.(2)Blood glucose and body weight After STZ injection, blood glucose levels ofall time points in the DM group were significantly higher than those in the control goup respectively(P<0.001). There was no significant difference in the body weight between the DM and control groups before the STZ injection(P>0.05), but in the 10th week after STZ injection, the body weight of DM group was significantly lower than that of control group(P<0.001), the mean body weight of the DM group was 30.4% lower than that of the controlgroup.(3)rCGRPmRNA and CGRP expression In the 10th week after STZ injection,the relative amounts of rCGRPmRNA and CGRP in DM group were 0.49±0.23 and 0.030±0.011, which were significantly lower than those in the control group measured as 0.88±0.27 and 0.064±0.022 (P<0.001).(4)ICP In the 10th week after STZ injection, the basal ICP andneurostimulation-induced ICP of DM group(8.7±3.4 and 54.6±17.5cmH₂O) were significantly lower than those of control group(36.8±11.2 and 156.3±38.4cmH₂O)(P<0.001). The decreasing rates of basal ICP and neurostimulation-induced ICP were 76.4% and 65.1%.ConclusionModeling DM rats with STZ(60mg/kg, intraperitoneally injection) is convenient and of high achievement ratio. The basal ICP and neurostimulation-induced ICP of the model are remarkably lower than those of normal healthy rats, therefore the model is fit to be animal model for DM erectile dysfunction study.Part III rCGRP gene transfer to penile corpora cavernosa of DM rats and its expression and effect on erectile functionObjectiveIt is to transfer pcDNA3.1/rCGRP to the penile corpora cavernosa and investigate its expression and effect on erectile function in DM rat.Methods119 male DM rats were randomly divided into three groups, group A treated with pcDNA3.1(+)/rCGRP, group B with nude plasmid of pcDNA3.1, and group C with PBS of 20% sugar. At 1st, 3nd, 7th and 14th day after the treatment, 9-10 rats were randomly selected from each group and determined for ICP, and then were killed and their penises were resected for testing rCGRPmRNA and CGRP expression with RT-PCR and western-blot.Results(1)GRPmRNA expression The relative amounts of rCGRPmRNA in group Awere significantly higher than those in group B and group C at 1d, 3d, 7d and 14d respectively(F<0.01, 0.001), but there were no significant differences in the amounts between group B and C(P>0.05). Within group A, B and C, there were no significant differences in the rCGRPmRNA amounts among 1d, 3d, 7d and 14d (P>0.05).(2)CGRP expression The relative amounts of CGRP in group A weresignificantly higher than those in group B and group C at 1d, 3d, 7d and 14d respectively (P<0.01, 0.001), but there were no significant differences in the amounts between group B and C(P>0.05). Within group A, B and C, there were no significant differences in the CGRP amounts among 1d, 3d, 7d and 14d (P>0.05).(3) ICP â‘ basal ICP There were no significant differences in the basal ICPamong the three groups at 1d, 3d, 7d, 14d respectively(P>0.05). Within group A, B and C, the basal ICP of 1d was significantly higher than those of3d, 7d and 14d respectively(P<0.05). â‘¡ neurostimulation-induced ICP Theneuro-stimulation-induced ICP of group A were significantly higher than those of group B and C at 3d, 7d and 14d(P<0.001), but there were no significant differences among the three groups at 1d(P>0.05). There were nosignificant differences in the neurostimulation-induced ICP between group B and C at 1d, 3d, 7d and 14d repectively(P>0.05). When compared in the neurostimulation-induced ICP among the four time points within groups, the ICP of 1d was significantly lower than those of 3d, 7d and 14d(P<0.01, 0.001), but the differences were not significant among the 3d, 7d and 14d in group A(P>0.05). There were no significant differences in neurostimulation-induced ICP among 1d, 3d, 7d and 14d in group B and C(P>0.05).ConclusionThe pcDNA3.1(+)/rCGRP is successfully transferred to the penile corpora cavernosa in DM rat, and it expresses rCGRPmRNA and CGRP continuously and stably for at least two weeks. The gene transfer can remarkably improve the erectile function of DM rats.