| Background and Purpose:Stress urinary incontinence(SUI) is a common disease in older women. It’s reported that the incidence rate was 27.6% in the older women,further it affected seriously the quality of life for the majority of women. It has caused the academic attention in Obstetrics and Gynecology that SUI is closely related to the changes in structural of the pelvics in female.Surgery is a common method of the treatment of SUI and suspension surgery has become the first-line surigical treatment. At present, the sling material in clinical contains synthetic polymers, the natural extracellular matrix material and acellular matrix derivative, for example, bladder extracellular matrix, pig leather and small intestinal submucosa, etc. The ideal material of the pelvic floor repairing should be safe, durable and biocompatible scaffold for cells seeded on the adhesion and growth. It is not used widely in the clinic, because that synthetic material has disadvantage of strong organizational erosion and scarring forming. The acellular matrix has been widely used in tissue engineering and regenerative medicine due to that it retains intracellular processing of tissue integrity and low immunogenicity. As one of the acellular matrix, reliminary studies indicates that UBM has the potential in the treatment of SUI, but the biggest problem is that it is inable to control effectively the degradation of the material and resulted the SUI in relapse. In recent years, with the further development of stem cell research and bio-engineering technology, some scholars has proposed that the transplantation of stem cell may be an effective means of the treatment of SUI, the experiment in animals has opened up the new directions for the treatment of SUI. The bone marrow mesenchymal stem cells could differentiate into smooth muscle cells and endothelial cells under certain conditions, so as to achieve the purpose of tissue repairing. The UBM loaded BMSCs has the low proliferation and differentiation into smooth muscle cells, which restricts the clinical application. It is the focus of researchers that how to improve the proliferation of BMSCs and differentiation into smooth muscle cells.Recent studies discovered that Laminin protein has the ability to promote adult stem cell proliferation and differentiation into a variety of cells, and the base layer on UBM also containes Laminin protein, but there is no relevant research about that the laminin protein on UBM material could induce BMSCs differentiate into smooth muscle cells.So this study was to investigate that the laminin on urinary bladder matrix influence the proliferation of bone marrow mesenchymal stem cells and the differention of BMSCs into smooth muscle cells, and provide a theoretical basis for building a new type of biological material for SUI.Experimental methods1. Preparation of BMSCsUsing the whole bone marrow adherent way in vitro isolation, culture and amplification of BMSCs in female SD rats, gestational age was 3-4 weeks, identified the immune phenotype of BMSCs by the flow cytometry phenotype, the P3 generation BMSCs as seeded cells.1.2 Preparation of UBMUsing mechanical separation and enzymatic digestion methods for processing fresh pig bladder, achieving the purpose of acellular, then freeze-drying, sealing after sterilization, which conduct as the tissue engineering materials.1.3 Preparation the UBM compositesPreparing the Laminin complex UBM and Anti-Laminin complex UBM composite materials used protein incubation, which were detected by the immunohistochemistry methods.1.4 BMSCs activity detectionThe experimental group contained Laminin complex UBM, UBM and Anti-Laminin complex UBM materials. The third passage BMSCs were seeded in 96-well plates according to 3000 / well, 120 ul cell culture per well. Taking the culture supernatant at cultured 1d, 3d, 5d, 7d, 9d, which were compared by LDH detection.1.5 BMSCs proliferation detectionExperimental groups were divided into three groups, such as Laminin complex UBM group, UBM group and Anti-Laminin complex UBM group. Each group had five holes, each hole had 3000 BMSCs seeded in 96-well plates. The cultured tissues were collected at 1d, 3d, 5d, 7d, 9d, 12 d, 14 d. The cell proliferation detection were compared by CCK8 detection.1.6 HE staining observation BMSCs migrationThe third passsge BMSCs were seeded into three groups of material according to 1X105/cm2, regular observation the medium. The tissue were collected at 3d, 5d, 7d, 14 d, using formaldehyde fixing, paraffin-embedded and slicing, The HE staining was used to observe the migration of BMSCs on different materials.1.7 Immunohistochemistry method observed the differentiation of BMSCs into smooth muscle cellsThe the fourteen day cultured tissue was collected, detected by immunohistochemistry method, The α-SMA of BMSCs was the direction of differentiation into smooth muscle cells.1.8 Statistical analysisUsing SPSS17.0 statistical software, measurement data used x ± s, that two groups were compared by anova.2 Results2.1 BMSCs isolation, culture and identificationMicroscope observation showed that primary BMSCs isolated grow adherently and swirlingly, had some miscellaneous cells,which could fuse up to 90% when were cultured one week. After underground passage, the cells were more uniform, regular shape and fast proliferation. Flow cytometry detection the expression of BMSCs showed that the positive rate of mesodermal origin molecule CD29 was 98.0%, expressing endothelial cell characteristics molecule CD31, the positive rate was 4.56%, the expression of hematopoietic stem cells characterized molecule CD34, the positive rate was 3.30%, in line with the BMSCs feature.2.2 UBM preparationVisually observation UBM material was dense and homogeneous translucent film-like tissue, microscopic observation showed that UBM was composed of dense and homogeneous collagen fibers, scanning electron microscope showed that homogeneous matrix composed of collagen fibers, collagen distribution uniform, the intact fibers and no residue cell.2.3 UBM composite preparationImmunohistochemistry methods showed that Laminin protein mainly expressed in the basal of UBM material, partly in non-basal. Laminin-UBM composite showed that Laminin protein expression increased significantly, Anti-Laminin-UBM group decreased significantly, indicating that the protein on the UBM can be enclosed by an antibody enclosed material.2.4 LDH detection BMSCs activityLDH analysis showed BMSCs could grow efficiently on the three materials, LDH value was increased significantly after the first 7 day cultured, especially when seeded on Anti-Laminin-UBM group, comparing with other two groups, 7-9 days cultured LDH curve reached parallel trend.2.5 CCK8 detection BMSCs proferationCCK8 analysis showed that BMSCs could grow efficiently on the three materials and had similar cell growth curve. BMSCs growed slowly in one week of culture, because the cells may be in a latent adaptation period, logarithmic growth after culturing one week, especially when seeded on Laminin-UBM composite,compared with other two groups.2.6 HE staining detection BMSCs migrationHE staining showed that three groups of material can support the growth of cell, adhesion, at the third day cultured, cell density raised significantly in the surface layers of Laminin-UBM composite than other two groups, with the inoculation prolonged, it was obviously that cells migrated to the deep of material, at the fourteen day cultured, some cells reached the bottom of material, the cell shape changed and the nucleus stretched to extend. At UBM group, only part of the cells migrated to the deep of material. While the cells seeded on the Anti-Laminin-UBM composite growed in the surface layers of material, and remained in the material surface with the inoculation prolonged.2.7 Immunohistochemical detection α-SMA of BMSCsImmunohistochemistry method detected α-SMA expression of BMSCs showed that the cells seeded on the Laminin-UBM composite was significantly positive, UBM group was weakly positive and the Anti-Laminin composite group was negative.Conclusion1. Successfully preparing UBM material;2. Isolation high purity of BMSCs;3. Building Laminin-UBM and Anti-Laminin UBM composite materials;4. It’s conformed that the Laminin protein on urinary bladder materialix have the ability to promote the proliferation, migration of BMSCs and the differention of BMSCs into smooth muscle cells. |
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