Combined Muscle Derived Stem Cells With Nerve Growth Factor Transplantation For The Treatment Of Female Stress Urinary Incontinence

Background:Muscle-derived stem cells(MDSCs)are currently the most suitable materials for the treatment of(Stress urinary incontinence,SUI),which can repair the damaged urethral sphincter complex.Nerve growth factor(NGF)can repair nerve and blood vessels and promote differentiation of stem cell.The construction of recombinant MDSCs cells with high expression of NGF gene as injection materials is a new idea for the treatment of SUI.Purpose:1.The recombinant cells were prepared by adenovirus vector mediated NGF gene transfection with MDSCs,the biological effects(cell activity,cytotoxicity,etc.)and the expression of the target gene were detected in vitro,so as to evaluate the feasibility of this cell as a urethral filler for the treatment of SUI.2.An animal model of SUI in rats was established to verify the recovery effect by using MDSCs modified with NGF.The mechanism of the engineered stem cells on SUI was also determined.The urinary control condition and the change of the structure of the urethral sphincter complex were also evaluated through urodynamic tests and histological analysis.Mehtods:1.MDSCs were isolated,cultured and identified in vitro.The adenoviral vector system was utilized to introduce NGF gene into MDSCs.Expression of NGF and its downstream target gene were detected by real-time quantitative PCR(qPCR).EGFP marker was used to determine the expression of NGF after transection.The biosafety and cell stability of the recombinant cells were also analyzed.2.A model of SUI in female rats was established by dissociating bilateral pudendal nerves.The successful animals model were randomly divided into 5 groups,sham surgery group,PBS only treatment group,MDSCs modified with NGF treatment group,no-load adenovirus transfected MDSCs treatment group and MDSCs treatment group.The urine leakage point pressure test was conducted at different time periods as the plan.After the last measurement,the animals were sacrified,and the urethral tissues were collected and stained with HE and Masson staining respectively to observe the changes in the micromorphological structure of the urethral sphincter complex.Furthermore,the Western Blot was used to detect the changes of NGF and VEGF.Results:1.MDSCs were infected with adenovirus at a multiplicity of infection(MOI)of 1000 with optimal expression efficiency of 80%.EGFP marker was observed at 72 hours after transfection and then gradually enhanced.NGF gene was highly expressed in the transfected MDSCs.2.The effect and mechanism of injection of MDSCs modified with NGF treating the rat model SUI were determined.Compared with other groups,the LPP of the rats treated with recombinant MDSCs was significantly higher than that of the other groups at all time periods respectively(P<0.01).Moreover,LPP reached its peak in the second week after treatment,followed by a small decrease in the fourth week and then stabilized at a higher level until the end of follow-up.Compared with the sham surgery group,the no-load adenovirus transfected MDSCs treatment group and the MDSCs treatment group also showed a small increase in LPP respectively(P<0.01),but the level of LPP was significantly lower than that of the recombinant MDSCs with high NGF expression.There was no significant difference in LPP between PBS treatment group and sham surgery group after treatment(P>0.05).Pathological sections of the urethra showed that after the treatment with recombinant MDSCs,the thickness and number of sphincter of the urethra were significantly higher than those in other treatment groups,which with less collagen deposition,cell debris and scar hyperplasia,and significantly more muscle fibers and vascular smooth muscle cells.The recombinant MDSCs treatment group showed the higher NGF and VEGF expression in urethral specimens.The no-load adenovirus transfected MDSCs treatment group and the MDSCs treatment group also showed a small increase compared with the sham surgery group(P<0.01).Conclusion:Adenovirus vector mediated transfection is safe and effective in engineering MDSCs.The NGF target gene can be introduced into the cells by adenovirus vector to obtain recombinant MDSCs with high NGF expression,without changing the cellular activity of MDSCs.The recombinant MDSCs with high NGF expression can improve the urine control function of rats and regenerate the structural complex of urethral sphincter,which may be a new choice for the treatment of SUI.