Vitro Experimental Pig Bone Marrow Mesenchymal Stem Cells Induced Differentiation Of The Bladder Smooth Muscle Cells And Urothelial Cells

Part1The cultivation and identification of BMSCs from Diannan Small-ear Pig in vitroObjective To acquire the method of cultivating and amplifying the bone marrow mesenchymal stem cells from Diannan Small-ear Pig in vitro, and to look for a new source of seed cell for the formulation of tissue engineering bladder.Method The Bone marrow mesenchymal stem cells were extracted from Diannan Small-ear Pig and were cultivated with adherent culture method after centrifugation. The morphological characteristics, growth and proliferation of the cells were observed. We use the method of MTT to describe and analyze the cells'growth. The CD29, CD44, CD45markers of BMSCs in the first and the second generation were examined by flow cytometry.Results The cells we cultivated were spindle-shaped in morphology. Flow cytometry results:P,CD29positive rate was99.44%, P,CD44positive rate was99.96%, P,CD45negative rate was99.55%. P2CD29positive rate was100%, P2CD44positive rate was100%, P2CD45negative rate was99.89%.Conclusion we can acquire BMSCs of Diannan Small-ear Pig by adherent culture method after centrifugation effectively. The second generation of BMSCs with high purity can be regarded as the source of seed cells. Objective:To explore the methods of primary cultivation, amplification and identification of smooth muscle cells and urothelial epithelia from the bladder of Diannan Small-ear Pig and to provide the seed cells for the construction of tissue engineering bladder. Methods:We performed the surgical operation to take bladder tissues of diannan small-ear pigs under the sterile condition. After the process of mechanical disintegration, we obtained the tissue of the mucous membrane layer and smooth muscle layer. At first, we acquired the bladder smooth muscle cells with the mixed enzyme method and the high purity urinary bladder epithelial cells with the trypsin digestion method. Then we cultivate the smooth muscle cells in DMEM/F12which contains10%fetal calf serum and cultivate the urinary bladder epithelial cells in DK-SFM(with epidermal growth factor and insulin). Morphological changes, growth and proliferation of cells were observed. Finally, smooth muscle cells and urothelial cells were identified through the methods of H-E staining and immunohistochemistry staining.Results:A great number of smooth muscle cells became adherent after12hours of primary cultivation and90%of them fused after2-3days. However, a large number of epithelial cells became adherent and appeared morphological changes after18hours.4or5days later,80%-90%of the primary cells fused. Urothelial cells appearing a typical "cobblestone"-like structure under inverted phase contrast microscope but smooth muscle cells showing a typical "valley and peak" form. H-E staining indicated that these cells were consistent with smooth muscle cells and urothelial cells. The results was proved to positive by immunohistochemistry staining.Conclusion:We can construct a cultivation system of cultivating smooth muscle cells and urothelia cells of pig's bladder in vitro.The cells we acquired through this system carried high purity and strong activity and can be used as the seed cells of tissue engineering bladder. Objectives To explore the method of inducing BMSCs differentiate to urothelial epithelia and smooth muscle cells(SMCs) in vitro, and to look for a new source of seed cells for the construction of tissue engineering bladder. Methods We selected the3rd and4th generations of BMSCs, urothelial epithelium and SMCs, and divided them into experimental groups and control groups. Experimental groups:Group A:BMSCs+urothelial epithelium+epidermal growth factor(EGF). Group B:BMSCs+urothelial epithelium conditioned culture solution+EGF. Group C:BMSCs+SMCs+basic fibroblast growth factor (bFGF). Group D: BMSCs+urothelial epithelium conditioned culture solution+bFGF. Control groups: Group E:BMSCs+EGF. Group F:BMSCs+bFGF.Group G:BMSCs2mL. The total time of common culture was about10days.we observed the cells'morphological transformation and took pictures for them after3,5,7,1Odays.The experimental groups and control groups were identified by smooth muscle actin antibodies and monoclonal antibody of keratin AE1/AE3.Results The cells'morphological transformation appeared after5days of induction in experimental groups. A large number of cells had transformed after7days obviously. About90%of the smooth muscle cells and urothelial epithelium had transformed into cells shaped like BMSCs in10days. However, there are no obviously morphological variation in the control group. Identifying with monoclonal antibody of keratin AE1/AE3,the keratin AE1/AE3was positive in group A and B, while negative in group E and G. Identifying with smooth muscle actin antibodies, the SMC actin was positive in group C and D, while negative in group E and G.Conclusions BMSCs have multi-directional differentiation ability. BMSCs which can be differentiated into urothelial epithelia and SMCs by simulating the micro-environment in vitro will provided a new source of seed cell for the construction of tissue engineering bladder.