Study On Myogenic Differentiation Of Rat Bone Marrow Mesenchymal Stem Cells Induced By Co-culture System In Vitro

Female pelvic floor dysfunction(PFD)is a group of diseases caused by weak pelvic floor supporting structures and injuries,including stress urinary incontinence(SUI)and pelvic organ prolapse(POP).The pelvic floor support structure is mainly composed of muscles,ligaments,fascia and nerves,and is connected to a complex,dynamic and coordinated system to protect the pelvic organs,providing basic guarantees for urinary function,defecation function,sexual function and other functions.Damage to any part of the pelvic floor will result in impaired function,and a series of clinical symptoms will appear.As the population ages,the incidence of pelvic floor diseases is expected to rise dramatically.Damage to muscle fibers is considered to be the first and most important cause of pelvic floor dysfunction,because its intensity weakens,causing displacement of pelvic organs and functional abnormalities.There are two kinds of treatment for PFD,non-surgical treatment and surgical treatment,all have their inadequacies,therefore,a new method is needed to restore the normal structure and function of the damaged pelvic floor muscles.With the development of tissue engineering,new methods are provided for pelvic floor muscle repair.Bone marrow mesenchymal stem cells(BMSCs)have two important properties:self-replicating and ability to differentiate into multiple cell lines,such as bone cells,chondrocytes,and myocytes,becoming tissue engineering.Seed cells,and easy to obtain,these characteristics provide a theoretical basis for the repair of pelvic floor muscles,treatment of PFD.ObjectiveTo explore how to induce BMSCs myogenic differentiation under in vitro conditions.Therefore,in this experiment,we used the method of indirect co-culture with normal levator ani muscle satellite cells and mechanically stretched levator ani muscle satellite cells to study the in vitro induced bone marrow differentiation of BMSCs.Methodology(1)Rat BMSCs were obtained by density gradient centrifugation and purified and subcultured.The morphology of BMSCs was detected by observing cell morphology and flow cytometry.And induce its differentiation into osteoblasts to prove its differentiation potential.(2)The levator ani muscle satellite cells were obtained by enzymatic digestion,purified and subcultured,and identified by observation of cell morphology and detection of Desmin by conventional immunofluorescence.(3)Take 3rd generation 80% fusion rat levator ani satellite cells,respectively,loaded 10% deformation,1Hz unidirectional horizontal stretching stimulation 0,6,12 h.The levator ani muscle satellite cytoskeleton was stretched at different times by phalloidin staining.(4)Rat BMSCs were indirectly co-cultured with levator levator ani satellite cells 12 hours after undistrection and distraction.Real-time quantitative RT-PCR was used to detect the expression of Myo D and Desmin mRNA in BMSCs.Western-blot was used to detect the expression of Myo D and Desmin proteins in BMSCs.SPSS 21.0 software was used for data analysis and processing,and the data were expressed as x±s.One-way ANOVA was used to test the difference between the samples and the control group,with ?=0.05 as the test level.Results(1)The fourth-generation BMSCs are mostly vortex-like or parallel-arranged and their morphology is relatively uniform.CD34(1.8%)and CD45(3.7%)molecules were negative and CD44(98.8%)and CD90(99.0%)were positive.And the resulting cells can be induced into osteoblasts.(2)The obtained rat pelvic floor levator ani satellite cell body is slender,spindle-shaped,uniform in size and shape,arranged in an orderly manner,and has strong refraction.The cells were cultured to the third passage and the Desmin immunofluorescence test showed positive expression,which was in accordance with the rat pelvic floor levator ani satellite cell characteristics.(3)The cytoskeleton of the stained satellites was observed under a microscope.The results showed that actin fibers(F-actin)were strongly red fluorescent stained and distributed uniformly.After mechanical stretching,the red fluorescence distribution of F-actin was still uniform and tidy,but the intensity was weakened.With the extension of mechanical stretching time,the red fluorescence intensity weakened more significantly,and the cells became narrow and long.(4)RT-PCR and Western-blot were used to detect the expression of MyoD and Desmin mRNA and protein in BMSCs from different co-culture systems.The results showed that compared with the control group,MyoD and Desmin of BMSCs in co-culture group and mechanical stretch co-culture group The expression of m RNA and protein were both increased(P <0.05).Compared with the co-culture group,the expression of Myo D and Desmin mRNA and protein in BMSCs was increased in the mechanical stretch co-culture group(P <0.05).Conclusions(1)Indirect co-culture with normal pelvic floor levator ani satellite cells and mechanically stretched pelvic floor levator ani muscle satellite cells can induce myogenic differentiation of BMSCs.(2)Indirect co-culture with mechanically stretched pelvic floor of levator ani muscle cells induces a stronger effect on myogenic differentiation of BMSCs.