Experimental Study On Phenotype Transformation Of Post-BOO Bladder Smooth Muscle Cell After Blockage By E2F Decoy Strategy

Bladder outlet obstruction(BOO) is the main cause of a series of clinic symptoms such as dysuresia , incontinence and renal failure, etc. Even if the cause is removed (eg. Relieving obstruction), bladder dysfunction persists in 35-45% patients. Bladder function in these patients cannot recover completely, furthermore, some got even worse. According our earlier works, the micro-structural basis of the phenomenon was the remodeling of SMCs(phenotype transformation) under the recurrent mechanical stretch beyond physiological endurance. Contractile type of normal SMC was transformed into proliferative type after overloading mechanical stretch.Detrusor muscle cells, one of smooth muscle cells(SMCs), are pluripotent , and have the ability to remodel to adapt the changing of outer environments. SMCs, which are widely located in many organs and different in appearance and function, cooperate with organs, tissues and other cells to achieve their function in vivo. In general, SMCs are distinguished as proliferative type and contractile type. SMCs of contractile type, mature and fusiform, have a strong power for contracting. On the other hand, SMCs of proliferative type were always found on early physiological stage or in pathological situation, which were more proliferative but less contractible. SM-α-actin, PCNA and cdk2 kinase were the signals of SMCs' phenotype transformation. Phenotype transformation from contractile to proliferative can be detected by the decline of expressions of SM-a-actin or the increase of expressions of PCNA and cdk2 kinase.E2F factor plays an important role in cell proliferation and differentiation. E2F factor, one of the most important transcriptional factors, connects with the binding site of cis element in late G₁ phase to activate the genetic expressions of c-myc, PCNA, cdk2 kinase downstream and phenotype converting of SMCs. Transcriptional factor decoy(TFD)oligodeoxyribonucleotide(ODN), artificial synthetic double-stranded ODN, has the same sequence of cis element. If TFD ODN is transfected , it may inhibit the activities of E2F factor by means of binding E2F factor and preventing its combination with cis element, which results in decline of related genetic expressions.For these reasons, three progressively experimental researches below were performed. First, by the application of cyclic mechanical stretch on the cultured DSMC, a new BOO model at cell level was established, which was based on the urodynamic effect of BOO on detrusor. Gradually phenotype transformation(cell remodeling) by mechanical stretch was studied on it; Second , transfecting E2F decoy ODN into SMCs before stretch to study the preventive effects for transformation by E2F decoy; Third, transfecting E2F decoy ODN into SMCs after stretch to study the reverse effects for transformation by E2F decoy.Main results and conclusions:1. Recurrent mechanical stretch applied to the cultured SMCs on the flexible-bottomed culture plate can be used to simulate the increase of urodynamic load experienced by detrusor under the BOO conditions. This is a brand-new method of experimental model of BOO at single cell level.2. Phenotype of SMCs was transformed from contractile to proliferative by mechanical stretch. MTT assay showed the significant increase of SMCs proliferation after mechanical stretch. Immunocytochemical stain displayed the down regulation of a-actin and disorder of cell form. Results of RT-PCR and Western-blotting showed the significant increase of mRNA expressions of PCNA and protein expressions of PCNA and cdk2 kinase by overloading mechanical stretch.3. Phenotype transformation of normal SMCs can be inhibited effectively by E2F decoy strategy by transfecting E2F decoy ODN before applying mechanical strech overload. MTT assay showed the significant decrease of cell proliferation. Results of RT-PCR and Western-blotting showed the significant decrease of mRNA expressions of PCNA and protein expressions of PCNA and cdk2 kinase by overloading mechanical stretch.4. Reverse phenotype transformation of proliferative SMCs may not be inducedcompletely by E2F decoy strategy by transfecting E2F decoy ODN after applying overloading mechanical stretch. Significant decrease of cell proliferation cannot be detected by MTT assay. Significant decrease of protein expressions of PCNA and cdk2 kinase was not found in Western-blotting results,either. Nevertheless, RT-PCR showed the significant decrease of mRNA of PCNA.