| Objective: To investigate the therapeutic effects and mechanisms of IGF-1 and IGFBP-3 combined with USCs transplantation on erectile dysfunction in diabetic rats by constructing diabetic ED rat model.Methods: A total of 90 SPF 2-month-old SD male rats were selected,weighing 230-280g(1 rat died during the 2-month feeding process).Fourteen rats were randomly selected as the normal control group(group A),and the other 75 rats were injected with streptozotocin(STZ)on an empty stomach.96 hours later,the random blood glucose of more than 16.7mmol/L was measured by tail vein blood sampling method as DM rats.We measured body weight and blood glucose on the day of STZ treatment and 2and 4 weeks after STZ treatment.Apmorphine(APO)erectile test was conducted at the fourth week after injection,and the remaining 69 rats were randomly divided into 5groups: diabetic control group(group B,13 rats),USCs treatment group(group C,14rats),LV-IGF-1 gene modified USCs treatment group(group D,13 rats).Lv-sh IGFBP-3 gene modification group(group E,14)and combined injection of LV-IGF-1 and LVsh IGFBP-3 modification group(group F,14).At the 2nd and 4th week after injection of different cell groups,6-7 rats from each group were randomly selected for erectile function test(ICP/MAP).After the test,the rats were sacrificed and their penile cavernous tissues were collected.Western blot was used to detect the protein expression of e NOS and α-SMA in the cavernous body,Masson staining was used to detect the smooth muscle/collagen fiber ratio of the cavernous body,and immunofluorescence was used to detect the expression of e NOS and α-SMA in the tissues.Results:1.Success rate of diabetic rats modeling: During the feeding period of one month after STZ injection,a total of 3 rats died due to hyperglycemia,and the blood glucose measurement of the tail vein of the rest rats was over 16.7mmol/L.The success rate of diabetic rats modeling was calculated as 72/75*100%=96%.2.APO results:After 1 month of successful modeling and feeding,all 72 surviving diabetic rats were injected subcutaneously with 1m L /kg APO solution in the neck.It was found that 3rats had obvious glans hyperemia and penis exposure,and the remaining 69 rats showed no erection.The calculated APO mold forming rate was 69/72*100%=95.8%.3. Erectile function test results: comparison between groups:(1)Compared with group A,the Max ICP/MAP measured in group B to F at week 2 and week 4 were significantly lower,and the difference was statistically significant(P<0.01).(2)Compared with group B,the Max ICP/MAP of rats in C to F groups increased significantly at week 2and 4,and the difference was statistically significant(P<0.01).(3)Compared with group C,the Max ICP/MAP of rats in groups D to F increased significantly at week 2and week 4,and the difference was statistically significant(P<0.01).(4)Compared with group D,Max ICP/MAP was slightly lower in group E at week 2 and 4,but the difference was not statistically significant(P > 0.05).The Max ICP/MAP of group F was significantly higher than that of group D at week 2(P<0.01),but there was no statistically significant difference at week 4 after treatment(P=0.177).(5)Compared with group E,Max ICP/MAP in group F increased significantly at week 2 and 4,with statistically significant difference(P<0.01).Intra-group comparison: After injection of different cell groups,Max ICP/MAP of each group in groups A to F showed no significant difference at week 2 and 4(P > 0.05).4.Western blot was used to detect the protein expression of e NOS and α-SMA in cavernous body of different experimental groups:The expressions of the two markers in the C～F treatment group were higher than those in the B group,but lower than those in the A group(P <0.05).Compared with C group,the expression level in D～F cell injection group was significantly increased(P < 0.05),and the expression level in F group was the highest among all cell treatment groups,but there was no significant difference between D and E groups(P > 0.05).5.Masson staining showed smooth muscle/collagen fiber ratio in cavernous body of different groups:The ratio of C to F group was much higher than that of B group,but lower than that of A group(P < 0.05).The ratio of D to F was significantly higher than that of group C,and the difference was significant in group F(P < 0.05),but there was no significant difference between group D and E(P > 0.05).6. Immunofluorescence showed the expression of e NOS and α-SMA in cavernous body of different groups:Compared with group A,the expressions of two markers in group B were decreased(P < 0.05),and those in groups C to F were significantly increased(P < 0.05).As for the comparison between group C to F,there was no significant difference in the positive cell ratio of e NOS(P > 0.05),while the positive cell ratio of α-SMA was the highest in group F and the lowest in group C(P < 0.05),and there was no significant difference between groups D and E(P > 0.05).Conclusions:1.Injection of urine-derived stem cells into the corpus cavernosum significantly improved erectile function in diabetic rats.2.After injecting LV-IGF-1 and LV-SHIGFBP-3 modified USCs respectively,erectile function was better than injecting USCs alone,and the combined injection had the best effect.The therapeutic effect of LV-IGF-1 was found to be superior to LV-sh IGFBP-3 in some mice,but there was no statistically significant difference between them.3.Urine-derived stem cells,IGF-1 and IGFBP-3 after gene silencing may promote erectile function recovery by improving vascular endothelial function and increasing smooth muscle/collagen fiber ratio. |
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