| Bone marrow mesenchymal stem cells (BMSCs) are the ideal cells for the treatment ofintrinsic sphincter deficiency (ISD) stress in urinary incontinence (SUI). In clinic, BMSCstransplantation around the urethral was used to treat urinary incontinence. With the bodytissue cells inducing, BMSCs differentiated into the corresponding adult cells to cure thedysfunction treatment of damaged muscle. However, so far, the therapy did not get theexpected effect. The reason was that little was known of the mechanism how BMSCs wasinduced into differentiation.And the key problem, which directly affected the stem cells inthe clinical therapeutic effect, was not solved yet.Previous studies indicated that BMSCs in the micro-environment of urinary bladdersmooth muscle cell in vitro can differentiated into smooth muscle cells. It prompted that thesignal path with which stem cells differentiated into certain kind of cells may exist in themicro-environment stem cells lives in.With the corresponding signal path, stem cells start agene-specific expression in time and space and the synthesis of specifict proteins.Thus, thecells changed in the structure, function and other aspects. The adaptability of stem cells tomicro-environment is the key to regulating the differentiation of stem cells into certain kindof cells. The research which studied the stem cells adaptability to the micro-environmenthas an important theoretical significance in understanding the nature of stem celldifferentiation.However, so far, the stem cell microenvironment adaptation mechanism isstill unclear.Consequentely, it has became very important to study the micro environment inducedadaptive regulation and mechanisms of stem cells in vitro and vivo. In recent years, thedifferentiation of histone acetylation modified stem cells has aroused widespread concern, Histone acetylation may be the significant incident of the cellular microenvironmentadaptability in important intracellular signaling regulatory. Acetylation covalent modifiednuclear histone was most closely related to the specific transcriptional regulation.The DNAdensity was regulated by the histone acetylation, which constituted a wide variety of"histone code". Generally, the chromatin remodeling was associated with transcriptionalactivation.On the other hand, the deacetylation of chromatin was associated with the genetranscriptional repression. Related inducible gene promoter in the vicinity of the histoneacetylation levels can affect a variety of biological transcriptional initiation. Thus, ineukaryotic cells, histone acetylation/deacetylation of chromatin structure and genetranscription regulation is a multi-layered, complex multi-step process.The cells in the experiments were separated from the3-weeks-old female SD rats bydensity gradient centrifugation and collagenase digestion. The simulation of pelvic floorenvironment in vivo constructed bladder smooth muscle in vitro contacting with BMSCsculture system.The establishment of BMSCs-to-SMCs differentiation model was designedto study the significance of histone acetylation in BMSCs differentiation into smoothmuscle and the role bladder smooth muscle microenvironment played in BMSCs adaptiveregulation mechanism. Here are the experimental methods and results:Identification of cell culture and common culture model1. Separation and cultivation of rat bone marrow mesenchymal stem cells (BMSCs)We took the bilateral femur and tibia of3-week-old female SD rats and repeatedly washedit with DMEM-F12medium bone marrow cavity and5ml injector.Mix the fluid, extractprecipitate with gradient density centrifugation, removed the supernatant and added theculture medium containing10%FBS DMEM-F12to cultural bottle and then culture at37℃,5%CO2incubator. We obtained the third generation of adherent cells and identify byflow cytometry molecular phenotype. Results: The successfully isolated bone marrowmesenchymal stem cells had a spindle shape.The molecular phenotype of cells was CD31,CD45, negative, CD29, CD44, positive.2.Isolation and cultivation of the rat bladder smooth muscle cells (BSMCs) Withsterile conditions BSMCs was obtained from3-week-old female SD rats’ bladder. Separate bladder smooth muscle cells with the collagenase digestion. Culture in10%FBSHG-DMEM and the resulting adherent cells were identified by immunofluorescence by theexpression of marker proteins. Results: The flag of smooth muscle gene a-SMA, calponinand SM-MHC are positive expression.3. Rat bladder smooth muscle microenvironment induced BMSCs differentiationmodel in vitro. The BMSCs and bladder smooth muscle cells were planted respectively ontwo sides of the suspension cell culture chamber transwell porous membrane (1um).Cellcultivation in six-well plates as the experimental group, no-co-cultured with the samegeneration as the control group the same batch BMSCs. After4days’ cultivation, detectsmooth muscle marker protein of experimental group and control with RT-PCR. WesternBlot and immunofluorescence. Results: RT-PCR results showed the experimental group hasa better landmark protein expression than the control group, indicating that BMSCs in thesmooth muscle microenvironment showed myoblast differentiation. It suggests that themodel achieve the goal.The effects of histone acetylation on the differentiation of BMSCs into BSMCs1. Micrococcus nuclease assay was done to test chromatin openness of smooth muscleiconic gene promoter region before and after BMSCs differentiation. BSMCs in passage3were seeded in the lower transwell chamber. After6h the transwell was turned over andcultured with smooth muscle broth. The next day, BMSCs were seeded on the interiorsurface of the membrane for3days. Then BMSCs PCR detection was done at different timepoints for micrococcus nuclease-treated experimental group and control group to test thethree iconic genes; α-SMA, calponin, SM-MHC promoter district chromatin openness.Quantitative analysis was done for DNA. PCR product using gel analysis software. Results:With the increment of time, the differentiation of BMSCs promoter region PCR efficiencywas significantly lower than the undifferentiated BMSCs. Contact with BMSCs co-culturecan significantly enhance the the BMSCs smooth muscle protein promoter region ofnuclease-sensitive, so that the promoter openness enhanced to open upstream resistancegene and expression of the downstream reporter gene, to increase the conversion efficiencyand the transformation frequency of the exogenous gene.2.ChIP was done to detect H4epigenetic state of α-SMA, SM-MHC and Calponinpromoter region. The BMSCs from upper transwell chamber in cultured group and control group were fixed with formaldehyde, interrupted into fragments of different sizes byultrasound, H4-specific antibody with a target protein to precipitate the protein-DNAcomplexes,65°C Solutions cross-linked complexes, separation and purification of DNA,after quantitative PCR amplification, amplification conditions were98°C for30s, withdenaturation at94°C for20seconds, annealing60°C1min, extension60°C1min,40cycles;smooth muscle iconic gene promoter acetylation differences before and after the Q-PCRdetection BMSCs differentiation. Results: After the co-culture, the degree of acetylation ofpromoter region in α-SMA, SM-MHC and Calponin had a significant increase comparedwith undifferentiated cells, it has been suggested that smooth muscle marker gene promoterregion DNA acetylation may be involved in the process of BMSCs differentiation intoSMCs.Conclusion: In this study, bone marrow mesenchymal stem cells and bladder smoothmuscle cells were successfully isolated, a co-culture system was also successfullyestablished and we simulated the vivo environment established vitro differentiation model.Based on the model, the effect of histone acetylation on the level of BMSCs differentiationhas been investigated. The study has shown that target genes promoter region exists a highexpression of acetylation in differentiated stem cells. |
PDF Full Text Request