| Research BackgroundErectile dysfunction (erectile dysfunction, ED) refers to sexual intercourse when the penis does not get an erection or can not maintain an erection to satisfy the sexual life, course of disease for more than 3 months. It is reported that 52% of 40 to 70 year-old male are disturbed by varied degrees of ED. There will be three hundred and twenty two million men suffered from ED until 2025 in world. ED has brought three major harms to patients:Mental disorders, psychosomatic pain; Reduce life satisfaction; Affect the family harmony, happiness and stability. Therefore, men with erectile dysfunction has been considered seriously by more and more experts, academics and the community.Penis is a special kind of vascular tissue. The normal penile corpus cavernosum is composited by Network-like cavernous trabecular and Trabecular space. Corpus cavernosum trabecular is composed by a large number of smooth muscle cells, during this period there are scattered extracellular collagen matrix, supplied by rich blood and nerve fibers in a relatively small component. There are many mutual communication between the trabecular sinus space, lined by the endothelial cells. Trabecular space formed by the confluence of cavernous sinus. Penile corpus cavernosum smooth muscle cells are the contraction and relaxation of the main ingredients.Normally, Corpus cavernosum smooth muscle cells not only have a rich adrenergic receptors, cholinergic receptors, but also contains a variety of non-adrenergic non-cholinergic receptors, as the main erectile tissue relaxation-contraction of components, it is the main undertaker of penile erectile function. In pathological conditions, the reduction of corpus cavernosum smooth muscle cells in penile erectile tissue is close to the incidence of ED, and with the same severity of clinical symptom. Many studies have found that the occurrence of ED is closely related to the reduction of corpus cavernosum corpus cavernosum smooth muscle cells in pathological state and the accumulation of collagen fibers. These pathological changes are inextricably linked to the corpus cavernosum smooth muscle cells in the pathological changes, because muscle is the key to the synthesis of collagen. More and more studies have found that corpus cavernosum smooth muscle cells may be directly involved in the starts and control of penis fibrosis when the ED occurs. In recent years, the research in the pathogenesis of ED goes deep into the molecular mechanism, for example, NO-cGMP pathway, ion channels, intercellular communication and the expression of various enzymes and receptors on the cell, etc. The materials which are studied in these molecular mechanisms are comed from the cultured corpus cavernosum smooth muscle cells in vitro, Christ and other scholars have studied the corpus cavernosum smooth muscle cells cultured in vitro by tissue sticking method, and have discussed usefully in the pathogenesis and treatment of the erectile dysfunction, but have not studied on rat corpus cavernosum smooth muscle cells in vitro by other ways, also a lack of analysing the cell purity of corpus cavernosum smooth muscle in vitro, and sufficient purity of the corpus cavernosum smooth muscle cells are a prerequisite for the study of erectile mechanism in the cellular level. In order to establish a high-grade penile corpus cavernosum smooth muscle cells in vitro, we successful establishment of a high purity of the original generation of corpus cavernosum smooth muscle cells through modified tissue explant, by differential adhesion and then purified passage, we could obtain a higher purity of the corpus cavernosum smooth muscle cells.Smooth muscle cell modulation refers to the change in mophorlogy, structure and fuction of smooth muscles under different stages body development or under different disease status. Smooth muscle cells differ from skeletal muscle cells and myocardial cells in the nature of differentiation. The differentiation of smooth muscle cells was reversible. And according to structure and function, smooth muscle cells can be further divided into two types:the contraction type (differentiated) and synthetic type (de-differentiated). The major fuction of the former one is contraction, and that of the latter one is proliferation, migration, secretion and regulation of extracellular proteins, etc. In the aortic tunica media, contain the following four markers:asmooth muscle actin (α-SMA), smooth muscle myosin heavy chain(SM-MHC), calponin 1 and h-caldesmon. Their expression are regulated by growth.α-SMA and SM-MHC are the major contractile protein which play a structural role, while h-caldesmon and calponin 1 are regulatory proteins that can act as markers for smooth muscle cells in a higher diferetiation stage. Osteopontin(OPN) is a negatively-charged extracellular matrix protein that is excreted by mesenchymal cells during development or under certain pathological conditions. The researches revealed that OPN is the marker for the changing of the phenotype of smooth muscle cells from the contraction type to the synthetic type, with the latter one expressing higher levels of OPN. Decrease in the expression of calponin 1 or increase in OPN demonstrate that the phenotype of smooth muscle cells modulated from the contraction type to the synthetic type.Corpus cavernosum is a special kind of vascular tissue as well. The research group has confirmed that the corpus cavernosum smooth muscle cell phenotypic transition exists in hypertension and diabetic erectile dysfunction rat through pre-experimental.As calponin 1 is the marker for contration-type smooth muscle cells and OPN for the synthetic type, we compared the expression levels of the relative expression levels of calponin 1 mRNA and OPN mRNA of the corpus cavernosum in rats of different groups in order to detect the phenotype modulation in the smooth muscle cells of corpus cavernosum in vitro.ObjectiveWe successful establishment of a high purity of the original generation of corpus cavernosum smooth muscle cells through modified tissue sticking, by differential adhesion and then purified passage, we could obtain a higher purity of the corpus cavernosum smooth muscle cells, as calponin 1 is the marker for contration-type smooth muscle cells and OPN for the synthetic type,to investigate phenotype modulation of corpus cavernosum smooth muscle cells cultured in vitro.Methods1.A total of 15 2-month-old SPF male rats were fed in the circadian rhythm of 12h, given tap water and high-protein diet while maintaining the space under a certain temperature and humidity.15 rats were divided into three groups randomly, that were Enzyme digestion group, Tissue sticking method group, and Modified tissue sticking group.The body weight were measured, subcutaneous injection of apomorphine (APO) (100μg/kg) was given, and the frequencies of penile erection in each rat in 30 min were observed and counted after administration, excluding the rats of erectile dysfunction. The penils of rat was separated carefully and cut into small pieces, these explants were seeded onto culture flanks and cultured in complete medium consisting of Dulbecco modified Eagle medium containing selected lots of 20%fetal calf serum and antibiotics at 37℃in a humidified atmosphere with 5%carbon dioxide.Cells growth was observed under phase contrast microscope Smooth muscle cell specific protein (a-SM-Actin and desmin) was identified by immunohistochemical methods. Statistical software SPSS 13.0 was used to analyze the data. All date were recorded in terms of mean±standard deviation ((?)±s) and the means were compared using one-wayANOVA and least significant difference (LSD) was used for multiple comparisons. There are significant statistical difference if the P value<0.05.2.Through the modified tissue sticking method we can obtain the original generation of corpus cavernosum smooth muscle cells,then through differential adhesion passage to the sixth generation, Leaving the primary cells, the second-generation cells, the fourth generation of cells and the sixth generation of cells for use. Calponin 1 is the marker of contractile smooth muscle and osteopontin (OPN) the synthetic smooth muscle. Quantitative polymerase chain reaction was used to analyze the relative expression of calponin 1 mRNA and OPN mRNA, and then the expressions of all the markers were compared. Use SPSS13.0 software was used to deal with the data according to the analysis of variance compared among means and least significant difference (LSD) was used for multiple comparisons. Since the variance of the expressions of calponin 1 mRNA and osteopontin mRNA among the four groups was irregular (F=5.431 P=0.009) and (F=5.131 P=0.011), Welch test statistic were used to analyze the approximate variance, and Tambane's T2 was used to conduct multiple comparisons. There are significant statistical difference if the P value<0.05.Results1.15 SD male rats are normal erectile function.2. Antisera directed against smooth muscle specific actin demonstrated positive immunostaining in both CCSM and fibroblast cell cultures,96.3% of smooth muscle, and 23.8% of fibroblast cells demonstrated positive immunostaining. Antisera against smooth muscle desmin demonstrated positively stained in the CCSM is 74.4%, and no staining of cells in fibroblast cell. Significant difference occurred in the positive cells rate for desmin among the three groups, the positive cells rate for desmin in modified tissue sticking group was remarkably higher than that in enzyme digestion group and tissue sticking method group. 3. Expression of calponin 1 mRNA of corpus cavernosum smooth muscle cells in the sixth generation group is the lowest in all four groups (F=337.342, P= 0.000) while highest in the primary group and the second generation group, there is no significant difference in expression of calponin 1 mRNA of corpus cavernosum smooth muscle cells between the primary group and the second generation group (P=0.072). Expression of OPN mRNA of corpus cavernosum smooth muscle cells in the sixth generation group is the highest in all four groups (F=125.572, P=0.000) while lowest in the primary group and the second generation group, there is no significant difference in expression of OPN mRNA of corpus cavernosum smooth muscle cells between the primary group and the second generation group (P=0.094).Conclusion1,Desmin can be served as specific monoclone mouse antibodies as markers for identifying the CCSM cells2,The more highly purified CCSM cell are obtained by modified tissue sticking culture.3,The architecture of smooth muscle to contract in corpus cavernosum is similar with that in vascular tissue.4,SD rat corpus cavernosum smooth muscle cell phenotype in vitro exists from the contractile to the synthetic transformation, CCSM is a very easy to divide the cells from a typical contractile phenotype into a synthetic phenotypewhen cultured in vitro to the four generations.5,Through the modified tissue sticking culture and the differential to be the second generation cells, we could obtain a higher purity and better functionality CCSM cells...
|
PDF Full Text Request