Experimental Study Of Muscle Derived Cell Autologous Transplantation For The Treatment Of Urethra Sphincter Injury Urinary Incontinence In Rat

Objective:Urinary incontinence (UI) is a urination functional disturbance disease , International continence society (ICS) definite that the patients uncontrol the urine flow outward in mind, there are more and more higher incidence rate with the aging increasing in our country. Though the UI have little effect on the patient's lives, but have a great effect on the quality of life. The UI incidence is three to forty percent in wen with trauma and post-prostatectomy by most statistics. The defects in structure and function of urethral sphincter is one of the key mechanisms in UI. Our body have myogenic effect, but it's limitted, so patients with ISD have myogenic-dominant damages with urethral sphincter. The treatments for wen UI include non-operation and operation, non-operation include drug treatment, electric stimulation, biofeedback, behavior therapy and so on, there havn't evident therapeutic effect. Surgical therapia method include: artificial urethral sphincter implantation, cavernous body of urethra ventrofixation, mucous membrane of urethra injection. But in earlier period the mucous membrane of urethra injection is one of the effect methods for UI because of ISD. Similar to female incontinence, the injectin therapy has the virtues of safety, convenience, micro-wound and has been welcome by doctors and patients. At present the common materials for injection, such as Teflon, collagen, body fat, silion microsphere,have evident side effect. The idea material should have some characteristics as following: integrateed construction, nonvenomous, no immunogenicity, no exclusion,longer effctive,having less inflammation in the site of injecting and no shifting from the injected site to other organs.With the development of cell tissue engineering ,people began to use cells with multipotential and plasticity characteristics to recover ,replace,repair impaired tissues or organs by injection therapy, and relative basic researches have been carried out extensively. In the process of studying the biological character of skeletal muscle cell, people find the skeletal muscle cell contain myoblast,the myoblast cell in adult muscle remain quiescent,but it can proliferate and fuse to myofibers to form new myofibers which repair the injury. In recently, one kind of muscle cell called muscle derived cell(MDC) was purified, which has very strong differentiative potential, it contains muscle satellite cell with stem cell properties.At present some researches reported MDC cardiac muscle injection has some benefits in improving the heart function for heart infarction.The lower urinary tract dysfunction,especially urinary incontinence ,the pathological field is small and no far to the surface,so MDC periurethral injection is easy.Comparing to other injectable agents,autologous tissue material has no problem of tissue compatibility and distant migration.In this study, we use the rat external urethral sphincter injuried model, muscle ederived cell autologous transplatation, observe the model urinary inconinence therapy.These lay a solid foundation for clinical using of autologous MDC periurethral injection in UI in future.Materials and Methods :1. Isolation, culture, purification, identification and transfection of MDC.We got gastrocnemius muscle of healthy adult Sprague-Dawley rats, and digested it with collagenase, dispase and trypsin. Preplate technique was applied to isolate MDC, the cell suspension was infunded in collagen-coated flasks.Preplat(PP1) represented a population of attaching cells within the first hour after isolation, PP2 within the next 2h, PP3 within the next 18h, and the subsequent preplates were obtained at 24h intervals(PP4-PP6). The preplate cells go through primary culture and serial subcultivation.The PP1-PP6 and differential myotube were identified by immunohistochemistry for a-Sarcomeric Actin.The second generation PP5,PP6 was marked with fluorchrome Hoechst 33258.2. Estabishment the animal model of UI and MDC autologous transplantationWe bulided the animal model of UI by cauterizing middle urethra and check the positive rate by urodynamic study and sneeze experiment. Then the UI animals were randomly divided into 2 groups: injection group (n=12), to obtain the MDC and tranfect Hoechst33258 then inject into periurethral tissue; sham injection group (n=12), to inject DMEM; control group (n=6), to expose urethra but no injection. Three rats were killed every groups in 1th,7th,14th, 30th day respectively to make urethra histological section and observe the injected cell' growth condition and tissue variation ; three rats of every group were studied by urodynamics after1week and 2 weeks.Result:1. The MDC was successfuelly purified and cultured by the preplate technique, PP1 and PP2 cells attached repidly, growed fastly , PP3 and PP4 belonged to satellite cells that attached slowly , PP5 and PP6 was MDC that attached most slowly. When primary culture for 48h,the PP6 cells keep round ,72h later,cells increased significantly and deformed long-fusiform cells.The third passaged cells proliferated well and were well-distributed.But in the 7th passage, cells proliferation slowed significantly, Serial subcultivation cells grow slowly and form evident myotube after differentiation.2. Immunohistochemical staining ofα—Sarcomeric Actin ,most of MDC cell have a positive result, from PPl to PP6 the positive rate was increased gradually,in myotube a-Sarcomeric Actin have a strong positive staining.3. The MDC were marked with Hoechst33258, we could see the living cell nucelus was blue, round, understain, regularity under fluorescence microscope, but the died cells were gathered and nuclear fragmentation. 4. The rats model of UI were estabished by cauterizing middle urethra. Urodynamic study for UI models showed LPP decreased significantly and evoking sneeze experiment is 89% positive.5. Differentiation of MDC marked with Hoechst33258 after injection.From the first to seventh day after injection, cells were small round and ellipse.They were distributed around the injected point. After 14 day, some high-fluorescent cells were elongated and occupied large area,but still localized within muscles surrounding urethra.In14 -30 days,some fluorescent cells were long-fusiform and had strong fluorescence, while some lost normal morphous,and then fluorescence weakened.6. Urodynamic study : abdominal leak point pressure( ALPP)in injection group,sham injection group,control group after injetion 1 week, three groups was : 20.48±1.08cmH2O,16.30±0.40cmH2O,24.46±0.35cmH2O respectively, (F=33.35, P<0.01), after two weeks three groups was: 22.36±0.64cmH2O,18.44±0.80cmH2O,25.44±0.58cmH2O respectively (F=59.58, P<0.01), The differences were statistically significant among three groups.This showed ALPP increased and evoking sneeze experiment decreased significantly after MDC injection.Conclusion:1. The preplate technique and enzyme digestion can isolate, culture and purify MDC.2.α-Sarcomeric Actin may identify MDC and myotube.3. Establish the animal model of UI by cauterizing middle urethra.4. Fluorchrome Hoechst 33258 may mark MDC.5. MDC autologous transplantation in earlier period can alive ,repair and replace injuried sphincter cells in rat, raise ALPP and improve control urine function significantly.