Dissertation > Medicine, health > Basic Medical > Human morphology > Human histology

The Experimental Study on Rat’s Adipose-derived Stem Cell Indirect Co-culture with Fibroblasts

Author ZhouMingHui
Tutor ZhangXiaoZuo
School Guangzhou Medical College
Course Obstetrics and Gynaecology
Keywords Pelvic floor dysfunction diseases Fat -derived stem cells Induced differentiation Fibroblast Co-culture Collagen
CLC R329
Type Master's thesis
Year 2011
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[Background] female pelvic floor dysfunction disease (pelvic floor dysfunction, PFD) pelvic support disorders caused by structural defects, damage and dysfunction, including stress urinary incontinence (stress urinary incontinence, SUI), pelvic organ prolapse (pelvic organ prolapse, POP), sexual dysfunction and fecal incontinence. According to statistics, women in the community in the United States, approximately 23.7% of the women there are one or more of the PFD and the prevalence increased with increasing age. The aging of the population also increased the PFD's disease burden ,2005-2006 for the treatment of the PFD economic costs over 1996-1997, an increase of about 1 times. Therefore, the PFD is a serious impact on women's quality of life, physical and mental health and to increase the people's burden of chronic diseases. Surgery is a common method of PFD therapy need women in the 80-year-old antecedents POP or SUI line harnesses the probability of surgical treatment was 11.1%. The modern concept of surgical pelvic organs to reach anatomical and functional recovery patch, patch include the synthetic patch biological patch. The single strand polypropylene mesh patch is considered to be the best synthetic material treatment PFD, but there are still some complications, including patch exposed to erosion, patch shrinkage caused due to the patch the higher incidence of pain and dyspareunia, up to 17%. The biological patch has good biocompatibility, compared with the synthetic patch, the incidence of complications related to the patch significantly reduced postoperative patch degradation problem has been the problem of the clinical application of biological patch. Collagen is the major part of the support structures such as ligaments and fascia, play an important role in maintaining pelvic support tissue elasticity and toughness. A number of studies show that female pelvic ligaments fascia fiber cell and cell-matrix type Ⅰ and Ⅲ collagen content decreases, suggesting a reduction in the amount of fibroblasts and collagen content lower is the PFD possible pathogenic mechanism. In 2003, Lee et al. Muscle-derived stem cells to treat rats with stress urinary incontinence, Yamamoto et al in 2010 reported two cases of the use of radical prostatectomy incontinence patients treated autologous adipose-derived stem cells (adipose-derived stem cells, ADSCs) are shows a good effect, suggesting the possibility of using stem cells for treatment of the PFD. Park et al found that The ADSCs can effectively promote collagen synthesis, as well as promoting fibroblast migration to the damaged tissue. So how do you promote ADSCs to the direction of the fibroblast differentiation and secrete more collagen? Reported, stem cells by co-culture with specific tissue cells can promote stem cell differentiation to specific direction. Therefore, the experimental contemplated by ADSCs with mouse embryo fibroblast co-culture, explore ADSCs and fibroblasts to synthesize and secrete collagen protein's ability to provide the experimental basis for ADSCs seed cells of the as a PFD biological treatment. [Objective] To explore ADSCs isolation and culture of experimental methods to study the biological characteristics of cell surface markers were observed to adipogenic, chondrogenic direction of multi-differentiation potential to provide experimental data for ADSCs experimental methodology. ADSCs and mouse embryo fibroblasts indirect co-culture, explore ADSCs and fibroblasts in promoting interaction between type Ⅰ and Ⅲ collagen synthesis ADSCs applied to pelvic floor dysfunction disease treatment organization. engineering experimental basis. 【Methods】 separation from the the SD rats groin fat pad, culture ADSCs were using MTT (methyl thiazolyl tetrazolium blue) method for cell growth assay, and cell cycle was detected by flow cytometry. 2 using flow cytometry the ADSCs cell surface markers: CD34, CD44, CD45, CD49d, CD90, CD105, and CD106. 3 applications into fat-inducing factor (dexamethasone, insulin, 1 - methyl-3 - isopropyl - xanthine and indomethacin) and into the cartilage inducing factor (TGF-β1, insulin, transferrin, and vitamin C) induced ADSCs adipogenic, chondrogenic differentiation. 4 ADSCs learn identification and mouse embryo fibroblasts indirect co-culture, the upper layer of the experimental group as fibroblasts, ADSCs were lower for; controls (1): The upper and lower layers are ADSCs were; control group (2): the upper and lower layers are as fibroblasts. (N = 6) Repeat hole, 6-well plates inoculated cell count of 10 to 5 / well, semi-quantitative RT-PCR was used to detect the different time points in each group I and III collagen mRNA expression. 5 detected by ELISA at different time points in each group of cell culture supernatant Ⅰ and type Ⅲ collagen content. [Results] 1 cultured in vitro proliferation of ADSCs rapidly showed a fibroblast-like growth, the cells were polygonal, uniform long spindle morphology; 1,3,5-generation cell growth curve of the \G0/G1 phase cells accounted for 90.6%. 2 in vitro the ADSCs surface antigen positive expression rate of: 99.88% of CD44 CD105 95%, CD90 99.74%-CD49d 89.56%, CD34 3.04% CD106 0.85%, CD45 0.26%; 3 in vitro ADSCs adipogenic induced three days after intracytoplasmic lipid droplets, and continue to increase, larger fusion induced 7 days after the part of the cell cytoplasm is completely filled with lipid droplets, induced 14 days after the oil red O staining orange. 4 cultured in vitro ADSCs into cartilage induction, the cell mass formation stability, after 14 days of induction, the diameter of the cell mass is increased to 2mm, smooth surface, Alcian blue staining. 5 semi-quantitative RT-PCR was used to detect the indirect co-culture cell collagen mRNA expression results: the experimental group lower ADSCs were collagen type I protein expression in co-cultured 6 days compared with the control group (1) lower ADSCs significantly higher, the difference was statistically significant; Ⅲ type collagen expression in co-cultured 6 days and 9 days compared with the control group (1), the difference was statistically significant. The experiments set of upper layer I and type III collagen protein expression of fibroblasts cultured for 3 days, 6 days and 9 days than those in the control group (2) the upper fibroblasts, a statistically significant difference. 6 ELISA detection of cultured cells the supernatant results show: indirect co-culture differences among the three groups of cell culture supernatants of type I collagen protein content in 6 days and 9 days, the difference was significant, the experimental group and the control group ( 2) the content to be higher (1), the difference was statistically significant, while the experimental group and the control group (2) the difference was not statistically significant; Ⅲ collagen content differ 3 days and 6 days in culture, The difference was statistically significant, the experimental group and the control group (2) the content of the control group (1), the difference was statistically significant, while the experimental group and the control group (2) the difference was not statistically significant. [Conclusion] an experiment successfully isolated and cultured ADSCs, its cell surface markers in line with the characteristics of ADSCs. ADSCs to adipogenic and chondrogenic differentiation, indicating that it has a multi-differentiation potential, can be used as seed cells in the pelvic floor defects bioengineering. 2 ADSCs mouse embryo fibroblast cells co-cultured cells secretion interactions raised I and type III collagen gene expression, the promotion of I and collagen type III protein secretion.

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