| Research BackgroundErectile dysfunction (ED) is a kind of common disease of Andrology, which trouble a lot of men.However, there is a lot of limitations in treatment, and the relationship between the changes tension in corpus cavernosum soomth muscle and erectile function has been recognized. Diabetes has been recognized as one of the most important risk factors for ED by most researchers, while ED has been a common complication of diabetes. The pathogenesis of how does diabetes lead to ED may be involved in many aspects, however, vascular factors is an important aspect. There is a structure of sinusoid in the corpus cavernosum,which is very similar with the vascular systems. Therefore, the corpus cavernosum is considered as a special kind of vascular tissue as well. Corpus cavernosum smooth muscle is the structural basis of cavernous space relaxing and penile erection, which plays a key role in the change of hemodynamics when the penis erects.Recent years, a lot of researches make the role of smooth muscle cells derived from cardiovascular more and more thorough. It is no doubt that vascular smooth muscle cells (VSMC) can be divided into two types according to its structure and function,: the contraction type (differentiated) and synthetic type (de-differentiated). The major function of the former one is contraction, and that of the latter one is proliferation, migration, secretion and regulation of extracellular proteins, etc. Some studies claim that the two types can be converted between each other, that is phenotype we put forward. Due to that differentiated VSMC can change into de-differentiated type when blood vessels are damaged or cultured vascular smooth muscle cells are stimulated by growth factors, it can be associated with corpus cavernosum, which is a special kind of vascular tissue, whose role similar to cardiovascular. It is unclear that the phenotype modulation in rats' corpus cavernosum soomth muscle cells with erectile dysfunction and its mechanism.In order to invest the phenotype modulation, we should distinguish the different state of smooth muscle cells'type. Different types have different markers, and the expressions of the type change varied with the phenotype. In the aortic tunica media, contain the following four markers:asmooth muscle actin (a-SMA), smooth muscle myosin heavy chain(SM-MHC), calponin 1 and h-caldesmon. We found that miR-145 can regulate the phenotype modulation of cardiovascular smooth muscle cells, and its expressions are associated with the state of differentiation after obtaining a great number of foreign literatures. In the period of embryo, we can test the expression of miR-145. Following the mature embryo, smooth muscle cells change to proliferation, and the expression of miR-145 decrease. The smooth muscle cells is in the state of differentiation after birth, and its expressions of the miR-145 increase. miR-145 can not only induced that pluripotent stem cells change into vascular smooth muscle cells, but also regulate the smooth muscle cells in the state of differentiation. The expressions of the miR-145 decrease in vascular injury and atherosclerosis, however, the proliferation of the smooth muscle cells is depressed and they change into the state of differentiation when the expression of miR-145 restore.Therefore, it is a new idea to investigate ED in phenotype modulation. miR-145 is a non-coding RNAs mainly exited in vascular smooth muscle cells, which regulate the proliferation and differentiation in many kinds of cells. We put forward that dose the regulation exit in the corpus cavernosum. In recent years, some researches reveled that miR-145 can inhabit the proliferation in vascular smooth muscle cells, and can make the state of proliferation change into contraction. There are no investigation in the corpus cavernosum smooth muscle cells.The expressions of miR-145 is very critical in the corpus cavernosum smooth muscle cell in diabetic rats with erectile dysfunction and the normal rats. Because the change of the expressions can indicate miR-145 may play important role in phenotype modulation in the corpus cavernosum smooth muscle cells in diabetic rats with erectile dysfunction.Basing on the studies mentioned above, we first verify the exit of phenotype modulation in the corpus cavernosum smooth muscle cell in diabetic rats with erectile dysfunction, then analysis the expressions of miR-145 in corpus cavernosum smooth muscle tissue. We establish the models of diabetic rats with erectile dysfunction, diabetic rats with normal erectile function, and norm rats. In 8 weeks, all of these rats have been fed in the same circumstance. Then we indicate that the phenotype exit in corpus cavernsum smooth muscle cell in diabetic rats with erectile dysfunction through testing the expression of the special proteins in the second cultured cells derived from the three models above by western-blot. We can also claim that miR-145 may regulate the phenotype in the corpus cavernosum smooth muscle tissue in diabetic rats with erectile dysfunction through testing the expression of miR-145 in the corpus cavernosum smooth muscle tissue in three groups above by qRT-PCR. ObjectiveIn order to understand the living habits and disease symptoms of diabetic rats with erectile dysfunction, we established the animal models including diabetic rats with erectile dysfunction, diabetic rats with normal erectile function, and norm rats. Three groups rats had raised in the same condition for 8 weeks. Then, to verify the existence of phenotype modulation, we tested the expressions of makers from the second cultured corpus cavernsum smooth muscle cells. In order to understand the role of miR-145 in phenotype modulation, we tested the expressions of miR-145 in the corpus cavernsum tissue throng qrt-PCR.Method1,26 8-week-old SPF male Sprague Dawley (SD) rats which were raised in the same room were divided into two groups randomly:(1) Diabetic group:14 rats, induced by a single ip of streptozotocin (STZ) (60mg/kg); (2) Control group:12 rats, were injected with an equivalent volume of citrate buffer. Take the random blood glucose level which was higher than 16.7 mmol/L as the criterion for successful diabetic animal model by measuring blood glucose at 72 hours,1 week and 2 weeks. There were 2 rats dying in the diabetic group in the 7th and 15th days after injected STZ.2,cell cultured The second-generation cells cultured which were fron the three groups of rats'corpus cavernsum were used for our research.3,cell identification We identified the smooth muscle cells through immunocytochemistry4,We tested the expressions of makers from the second cultured corpus cavernsum smooth muscle cells through western-blot. The steps of western blot are as follows: (1),Electrophoresis; (2),Transmembrane; (3),Incubation the first antibody;(4),Incubation the first antibody; (5),Exposuring.4,We tested the expressions of miR-145 in the corpus cavernsum tissue throng qrt-PCR. The steps of qrt-PCR are as follows:4.1,The extraction of total RNA; 4.2 Removing genome; 4.3 Testing the purity and integrity of total RNA; 4.4 RT; 4.5 Quantitative PCR.5,Analysis the strips of western-blot and the CT,ΔCT,ΔΔCT and 2-ΔΔCT from qrt-PCR and finding the change of the expressions of miR-145 from the different groups' corpus cavernsum tissue.Results1,The weigh and glucose of rats There were 2 rats dying in the diabetic group in the 7th and 15th days after injected STZ. The diabetic rats became polyuria, polydipsia and thin. The weigh of diabetic rats was decreased obviously comparing control group(P<0.01), and the glucose of diabetic rats was increased obviously comparing control group(P<0.01).2,The result of APO experiments. In control group, we deposed 2 rats that were negative in the APO experiment. In diabetic rat group, there are 7 positive rats and 5 negative rats.3,We identified the smooth muscle cells through immunocytochemistry, and the picture were showed in the paragraph.4,The result of western-blot We got the results of expressions in SMA and SMMHC through the brightness of the strips in western-blot.5,The results of qrt-PCR are as follows:the results are denoted by means±standard deviations. The value of CT is 16.32±0.77,ΔCT is 5.25±0.79, andΔΔCT is -3.69±0.78,2-ΔΔCt is 14.76±8.82 in control group. The value of CT is 16.31±0.34,,ΔCT is 5.39±0.44, andΔΔACT is-3.55±0.45,2-ΔΔCt is 12.11±3.31, in diabetic rats with erectlie dysfunction group. The value of CT is 18.03±0.66,ΔCT is 7.42±0.98, andΔACT is -1.53±0.99,2-ΔΔCt is 3.41±1.77,2ΔΔCt in diabetic rats with erectlie dysfunction group. Comparing the value of 2-ΔΔCt are as follows:Control group VS diabetic rats with rectile dysfunction(P<0.01),diabetic rats with erectle dysfunction VS diabetic rats with normal erectle function(P<0.05). |
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