| Intrinsic sphincter deficiency (ISD) is the main reason of stress urinary incontinence (SUI). Surgeries and nonoperative therapies are included in the treatment of SUI, these methods have changed the pelvic structure, but still cannot relieve patients from ISD, for the function of sphincter was not restored. In recent studies, people have found that the stem cell transplantation can be used in muscle injure recovery and muscle contract improvement. However, this therapy have not yet achieved the expected effect, for there is little knowledge on the post-transplantation differentiation of the stem cells, thus its differentiation capacity can not be completely used in clinical treatment. So to clarify the accommodation induced by both in-vitro and in-vivo microenvironment and its mechanism is becoming vital to our cause.In recent years the effects of histone modification on stem cell differentiation has captured people's attention. Histone modification is the covalent declaration of histone by includingacetylation and methylation, while not changing the cell's DNA sequence. These modifications served as markers in gene expression and produced synergistic or antagonistic effect in the process of combination between other proteins and DNA. These modifications are dynamic transcriptional controlling factors also known as histone codes. These codes can be identified by specific protein, then can be translated into specific chromatin state for the regulation of specific genes. In these codes, histone acetylation relates most closely to the regulation of gene transcription. It was reported that the acetylation of lysine residue on histone H3 subunit played an important role in chromatin packaging and genetranscription. Generally speaking, there is a close relationship between the acetylation of histone H3K9 and genetic activation. Conversely, H3K9 deacetylation was tied up to genetic silence. The DNA configuration was loosened in the existence of acetylation, then transcriptional factors could be combined to the corresponding cis-acting original body in nucleosomes, and in the mean time, corresponding trans-acting factors was recruited, then gene transcription can be started. DNA in the downstream area of the region in which the density of histone acetylation was high showed extraordinary transcriptional active. However would stem cell exhibit the same accommodation, under a certain microenvironment, through histone modification, as other environmental cells, is still unknown.Bone mesenchymal stem cells (BMSCs) is a small pack of no-hematopoietic cells in bone marrow, They have potentials in proliferation and multi-directional differentiation. They can be easily obtained, multiplied, and can be used in auto-transplantation, therefore, can be used as seeding cell in cell therapy. This study is based on female SD rats, we use density gradient centrifugation and collagenase in BSMCs and BMSCs culturing; then a in-vitro model was established to simulate the differentiation from BMSCs to bladder smooth muscle cell, using direct contact co-culture technique, in purpose of monitoring the histone acetylation level in the promoter of smooth muscle related gene during in-vitro smooth muscle cell differentiation and the effect of histone acetylation on the differentiation from BMSCs to BSMCs. The main results and conclusions were summarized as follows:Cell culture identification and co-cultured model establishment1. BMSCs separation and cultivation:use the ensity gradient centrifugation to separate cells and The positive rate of CD29,CD44,CD166 were 96.55%,92.96% and 99.54% respectively, while only 1.39% for CD31 and 5.56% for CD45 in the separated BMSCs in flow cytometric analysis.2. BSMCs separation and cultivation:use the method of collagenase digestion to separate cell, Immunofluorescence staining indicated thatα-SMA,calponin and SM-MHC were strong expression in BSMCs.3. Establish BSMCs and BMSCs co-cultivating model:In vitro use transwell porous membrane as BMSCs and BSMCs interval, BMSCs and BSMCs were vaccinated in porous membrane sides respectively for the test group, while the same batches of BMSCs co-culture without contact were set as control group. Take three days of contact co-culture, RT-PCR detection the smooth muscle differentiation landmark protein. Results:RT-PCR show that a-SMA,calponin and SM-MHC were stronger expression in BMSCs after co-culture than control group indicated that we success for setting mode.Histone acetylation influence to the differentiation of BSMCs1.The ChIP cluster generator attempts, smooth qPCR analysis landmark gene promoter area histone acetylation level on the influence of BMSCs to smooth muscle differentiation, tested by ChIP cluster generator attempts in a living cell technology under the condition of formaldehyde in BMSCs fixed before and after the training complex, protein-DNA fragments, adjust chromatin ultrasonic interrupted in 200-1000bp segment size, and then through the fight histone acetylation site specific H3K9 antibody precipitation quantitatively, BMSCs genome before differentiation in BMSCs PCR smooth muscle landmark gene promoter area DNA fragments, understand stem cells to differentiate the promoter region before and after the change of acetylation degree, thus obtains proteins and DNA interaction of information. Results:BMSCs smooth muscle landmark gene promoter area acetylation degree is undifferentiated increased significantly after co-culture, the promoter regions acetylation level was increased after BMSCs differentiation.2. Histone acetylation enzyme inhibitors (HDACi) sodium butyrate influence to the differentiation of BMSCs:Culture the second The second generation BSMCs on the transwell surface,we took the transwells to incubator room after 6 hours move which to the six orifices plate with SMCs culture liquid.The next day,we translated BMSCs to the other surface of transwell,the positive group was treatment with 3.0mmol/L sodium butyrate co-culture solution,then we took BMSCs which co-culture 1,2,3 days,cellular immunofluorescence staining and RT-PCR analysis that differentiation in different period. Results:HDACi sodium butyrate can strengthen BMSCs differentiation capacity, increase histone acetylation level can enhance BMSCs differentiation.Conclusion:In this study, we have BMSCs and BSMCs separated successfully and established a cell direct contact co-culture model. Then based on this model, the influence of histone acetylation on BMSCs differentiation was investigated. Our work showed that, after the differentiation of stem cells, the acethlation level increased significantly in the promoter of target gene; the differentiation ability of mesenchymal stem cells can be greatly increased by elevating the acetylation level in co-culture environment. In this study, we explored the regulatory mechanism of histone acetylation on the differentiation of mesenchymal stem cells. Thus laid down a foundation for clinical usage of BMSCs transplantation in SUI treatment. |
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