Effect And Mechanism Of Overexpression Of INOS In Rat Levator Ani Muscle Satellite Cells Into Muscle

[Background]Stress urinary incontinence(SUI)is a high incidence in women,which can cause serious physical and mental harm to the patients and bring huge economic burden to the society.Pregnancy and vaginal delivery are considered to be the main causes,and levator ani muscle injury is the main pathological change.In our former study,it has been proved that the levator ani muscle is impaired and the expression of nitric oxide synthase(iNOS)is reduced in SUI model rats,which may be involved in the development of stress urinary incontinence.The activation and differentiation of muscle satellite cells(SC)play an important role in the regeneration and repair of skeletal muscle injury.INOS is the rate limiting enzyme for the synthesis of nitric oxide,and whether the overexpression of iNOS in the levator ani muscle satellite cells can promote the formation of satellite cells to repair the injured levator ani muscle has not been confirmed.[Objective]To explore the effect and mechanism of overexpression of iNOS in rat Levator ani muscle satellite cells(SC)Into muscle,which the SC was isolated from SUI model rats Levator ani muscle and culture in vitro,then the SC was restructured by slow virus mediated iNOS.[Methods]The levator muscle tissue was got from SD-rat anus under aseptic condition,dealt with collagenase and trypsin enzyme digestion,400 mesh stainless steel screen separation.The SC was purified by using differential adherent method and the SC was identified using Pax7,MyoD,Desmin,alpha-SCA labeled with immunofluorescence.The identified SC was inoculated to 96 orifice plate using CCK-8 method to draw the growth curve,and expand the culture conditions in vitro passage.RNA extraction from the lung tissue of SD rats,two step reverse transcription cDNA was used and primers were designed with restriction sites of the full-length open reading frame of iNOS which was obtained from cDNA library.The recombinant plasmid of pLenti-C-mGFP-iNOS connection was constructed by double enzyme digestion and T4 DNA ligase,lentivirus(carrying the iNOS ORF base sequence)was obtained by the slow virus packaging kit.to package it.SC was infected by lentivirus to construct the stable overexpression of iNOS SC,GFP was positive under the fluorescence microscope.The normal levator muscle satellite cells was the control group,recombinant cells was the experimental group.mRNA and protein levels of activation marker MyoD,differentiation marker Desmin expression were tested and the myogenic phenotype was observed microscopically.[Results]By using collagenase and trypsin digestion,the satellite cells from the levator ani muscle of SD-rats can be successfully separated and purified by differential adhesion method.Satellite cells can be cultured and subcultured in vitro.The cells were amplified by multiple times,and the cell activity decreased after four to five passages.The overexpression of iNOS could be mediated by lentivirus,and the positive rate was over 60%.The MyoD and protein levels of mRNA and Desmin in the recombinant satellite cells were increased,and there was statistical difference compared with the control group.[Conclusion]The SC was isolated from the levator ani muscle of SD-rats and cultured in vitro successfully;The SC which was reconstructed by lentivirus showed more power into muscle.The study provide a probability to treat SUI using SC..