The Experimental Study Of Transplantation Of Human Skeletal Myoblasts On Fecal Incontinence

Background:Anal incontinence(fecal incontinence) is that the organism lose the ability to control fluid or solid content and gas in the rectal,resulting in the increasing frequence of defecation. Patient whose symptom is light presents that stool contaminates the shorts, and the severe presents the whole discharge of the content of rectal.Although it cannot threaten the patient's life,it brings serious physical and physiological pain and inconvenience of living to the patient,and seriously influence the quality of life of them.Because of insufficient congnition and inadequent methods of diagnosis,these problems are ignored so far.It is reported that about 84.8 percent of the patients are missed diagnosis.Presently, the incidence of anal incontinence in different populations is unequal.In Americal and Europe,the incidence is 2～7 percent,and increases because of aging and the decline of psychophysiological state.The mechanisms of feces control is followed:(1)Perception of oncotic pressure by anal activates the kinetic response of defecation control;(2)The tension of the internal sphincter muscle of anus makes it closed inmost of the time.(3)Tension of anal sphincter and puborectalis mantain the angle between rectum and anal canal and make anal canal closed during the relaxation of anal sphincter ;(4)adaptability of rectum makes rectum engorge in the cases of the not too high pressure of rectum .Destruction of any mechanism above will result in fecal incontinence .Nowadays treatment include etilogical treatment ,dietary therapy ,contract exercise of sphincter, mind-cure and biofeedback therapy ,etc.Cases of falure in internal medicine treatment need different operation to strenthen function of sphincter , or implantation artificial anal sphincter, but result of those treatment is not so good as expected .Some patients may need implant man-made anus at the cost of low living quality.As scientists go deep into the research of mechanism of self-control of anus ,the part of anal sphincter playing in self-control of anus is considered much more important than before .Anal sphincter is incrassation of colonic cricoid smooth muscle around anus, which takes 80% of the anal resting pressure ,as the anal resting prssure grades is one of the primary factors which manage self-control of anus at rest .It makes anal canal closed down in most case , especially of great meaning to self-control of gas and liquid.Research of ultramicro- structure of anal sphincter in the cases of patients with fecal incontinence shows : shortness of smooth muscle cells of anal sphincter , destruction of normal structure of the rest cells , elongation of elastic tissue and increase of collagen fibers. Thus the most familiar cause of copracrasia is Degeneration of finespun smooth muscle of anal sphincter which mantain anal sphincter closed .One of the pathomechanism of fecal incontinence is decrease of anal resting pressure . It will be great importance to control defecation with a posibility of reconstruction anal sphincter with normal physiologic function as smooth muscle tissue can not recovery itself after damaged.Objective:Skeletal myoblast is the precursor cell of muscle, also the dormant mscl-stem cell stem cell with the potency of multiplication and differentiation,which is between muscle serum coat and basal lamina of skeletal muscle fiber in the adult skeletal muscle.It firm to myotubule fromproliferate, differentiation, confluence,after skeletal muscle cell destroyed.Thus repairing the impaired myoideum. At present adult smooth muscle precursor cell uncertain yet. Sarcoblast my qua one smooth muscle precursor cell, is likely to treat copracrasia from rebuilding sphincter muscle of anus.Masses of study already indicate that human myoblast can survival in the rat of immune defect and immunosuppressive drug treated. Thus the experiment will cultivate human musculi skeleti myoblast. Transfection of Ad-GFP to myoblasts in vitro and detection transfection efficiency as well as it's contribution to the musculi skeleti myoblasts proliferate and differentiation.Studying in vitro, the transfection of Ad-GFP to myoblasts which Transplanted in the archo- smooth muscle of the immune defect rat. Observe surviva of transplant cell and effect on anus resting pressure.Methods:1. Isolation and culture of skeletal myoblastLong adductor muscle were obtained from the adult, then digested by trypsin and collagenase, finally monoclone in culture medium,observed under inverted microscope every day. The cultured cells were identified by immunofluorescence staining with desmin.And observed the influence on cryopreservation and resuscitation to myoblas proliferation and differentiated.2. Transfection of Ad-GFP to myoblasts and effects on proliferation and diferentiation of myoblasts. Human embryonic kidney(cells) casing, Ad amplification (Ad-GFP), virus titer determine. Infection with 100 titrations of Ad-GFP. Transfection efficiency of adenovirus vector to myoblasts were identified by Cytometry. Observed morphocytology alteration under inverted microscope. Detected the activity of cell proliferation with CCK-8. Finally, we also examined the diferentiation efect of Ad-GFP on myoblasts.3.Study of the transfection of Ad-GFP to myoblasts which Transplanted in the intrarectal of the immune defect rat.The immune defect rats Were randomized in to 2 groups( 10 in each group):The experimental group transplanted with myoblasts transfected by Ad-GFP; the control group injected PBS. All animals were followed 4 weeks after the operation.Survival and immigration of transplantated cells, were assessed by immunofluorescence staining, myoblast transplantation function was assessed by anus resting pressure and electromyogram.Results :1. The myoblasts derived from the adult human skeletal muscles were cloned and after the cryopreservation and resuscitation myoblasts still could keep the ability of proliferation and differeration .2. Myoblasts could be effectively transfected by Ad-GFP in vitro.Over 80% ofMyoblasts were transfected at MOI 100.And there was no significant diversity between myoblasts trabsfected with Ad - GFP and those without Ad - GFP (P>0.05),which proves labeling myoblasts with adenovirus expression vector containing green fluorescent protein is safe , easy and effective , and can remain its proper biological characteristics.3.Survival of the transplanted myoblasts was detected by pathological examinatio at 28 days.The green fluorescence was seen diffusely around the injection area,the myoblasts transplanted in rectal smooth muscle could servive ,and anal resting pressure was increased , the results of the treatment group were significantly superior to the control group(P<0.01),which means transplantion of myoblasts can improve the function of sphincter ani .Conclusions:1. Skeletal myoblasts can be successfully cultured in vitro2.Myoblasts could be infected with Ad-GFP safely and efficiently in vitro. The subsequent research could use the marker gene.3.Skeletal myoblasts cultured in vitro can survive in the transplanted region,raised anus resting pressure and myoelectricity swing, improved anal sphincter function.