The Experimental Study On Rat's Adipose-derived Stem Cell Indirect Co-culture With Fibroblasts

BackgroundFemale pelvic floor dysfunction(PFD) is caused by the support structure defects and damage, including stress urinary incontinence(SUI), pelvic organ prolapse(POP), sexual dysfunction and fecal incontinence. According to statistics, women in the United States'community there are more than 23.7% have one more diseases, and the prevalence increases with age. In 2005-2006, the costs for the PFD is up to 412 million U.S. dollars, about 2-fold in 1996-1997. So, the PFD is a serious chronic disease impact on women's quality of life and health.Surgery is the main method in the treatment of PFD. In a woman's lifetime,11.1%women were likely to have one more sling surgery becouse of SUI or POP.And the incidence is increases year by year in women's lifetime. Modern surgical concept is use tension-free mesh to recovery the pelvic floor's function and the anatomy, including synthetic mesh, biological patch. Monofilament,large pore Polypropylene mesh is considered the best synthetic material in the treatment of PFD, but it still has some complications, including the mesh exposed, erosion, shrinkage. Even more, the mesh related pain and sexual dysfunction is up to 63%. Biological mesh has good biocompatibility, as compared with the synthetic patch their patch. It can significantly reduced the incidence of related complications, but the degradation after surgery is obstruct its clinical application .Collagen is the support structures's main component, including ligaments and fascia. The ligaments and fascia are the main component of the pelvic floor, which play a important role in providding enough flexibility and tension to support the pevic organ. Many studies showed that the content of typeⅠ,tylpeⅢcollagen and the number of fibroblasts is decrease, suggested that the reduce of the amount of fibroblasts and collagen content is the possible pathogenic mechanism of PFD. 2003, Lee, et al.use muscle-derived stem cell therapy with stress urinary incontinence in rats. 2010, Yamamoto et al. reported the use of autologous ADSCs therapy with urinary incontinence in 2 cases, after radical treatment prostate cancer , have shown good results. Prompted that use stem cells therapy with PFD is possible. Can we use stem cells to treat POP? ADSCs can effectively promote collagen synthesis, and promoting fibroblast migration to the damaged tissue . So how do we make ADSCs secrete more collagen as well as how to promote the ADSCs differentiate to fibroblasts ? Some researchers reported, stem cells co-cultured with a specific cells can promote stem cell differentiation to the specific lineal cells. Therefore, we use ADSCs co-culture with embryos fibrolasts , hoping to improve the ADSCs and fibroblasts secrete more collagen,and promote ADSCs differentiate to fibroblast-like cells.Objective1.Establish a method of isolation and culture ADSCs in vitro ; observe the biological characteristics of the ADSCs; investigate the capablity of differentiating into adipocytes, chondrocytes, and osteoblasts.2.Use ADSCs indirect co-culture with embryonic fibroblasts , to discuss the effection of promotion typeⅠand typeⅢcollagen secretion, to investigate the feasibility of ADSCs as seed cells in treatment pelvic floor dysfunctional diseases.Materials and Methods1.The fat pad was obtained from the rat inguinal, and digested with collagenaseⅠto isolate ADSCs; use MTT detected the cell proliferation capablity in vitro; and the cells cycle were detected by flow cytometry; 2.Use flow cytometry detecting ADSCs'cell surface markers: CD34, CD44, CD45, CD49d, CD90, CD105, and CD106;3. Use adipogenic inducing factor (dexamethasone, insulin, 1-methyl-3-isopropyl-xanthine and indole Indomethacin), and chondrogenic inducing factor (TGF-β1, insulin, transferrin, and vitamin C) induced ADSCs differentiate into osteogenic, adipogenic,and chondrogenic cells;4. Use ADSCs indiret co-culture with embryonic fibroblasts, then use RT-PCR detected tylpeⅠand typeⅢcollagen gene expressiona at different time points;5. Use ELISA test the typeⅠand typeⅢcollagen protein secreted by ADSCs and fibroblasts at different time points.Results1. The ADSCs propagated rapidly in vitro, and constituted a homogenous fibroblast-like morphology, the cells shapes were polygonal,and spindle; the cell growth curve were "S "shape ; 90.6% ADSCs were G0/G1 phase cells.2. Flow cytometric analysis the cell surface markers demonstrated that: CD44 99.88%,CD105 95%,CD90 99.74%,CD49d 89.56%,CD34 3.04%,CD106 0.85%,CD45 0.26%.3. After adipogenic induced, intracellular lipid droplets could be observed, and the lipid droplets were fusion become bigger and bigger, the intracellular lipid droplets present jacinth after by Oil Red staining.4. After chondrogenic induced, the cells form a stable micromass; 14 days after induced, the micromass's diameter increased to 2mm, alcian blue staining was positive.5. RT-PCR results showed that co-cultured ADSCs'typeⅠcollagen gene expression were more than the control group at 6 days,and the difference was significant; typeⅢcollagen expression were more than the control group 6 days and 9 days, and the difference was significant. The fibroblasts'typeⅠand typeⅢcollagen gene expression ,after co-culture 3 days, 6 days and 9 days ,the experimental group were higer than control group, the difference was significant.6. Co-culture supernatant's collagen concentration detected by ELISA test showed that: supernatant of cell culture secretion of typeⅠcollagen in 6 days and 9 days are different, the difference was significant; cell culture supernatants of typeⅢcollagen secretion in cultured 3 days and 6 days are different, the difference was significant. Subsequent comparison between two independent samples found that the experimental group and control group 2 was no difference, but the experimental group and control group 2 were higher than control group 1, the difference was significant.Conclusion1.Our experiment successfully isolated ADSCs, the cell surface markers consistent with the characteristics of ADSCs.The study was successful induced ADSCs to adipogenic and chondrogenic cells, indicating that the ADSCs isolated from rat have the ability of multi-potential differentiation.ADSCs could be the seed cells in treatment PFD.2.The study revealed: ADSCs co-cultured with embryonic fibroblasts can promote typeⅠand typeⅢcollagen gene expression and protein secretion, by the interaction of cells.