Expressions of Type I Collagens in Rat Adipose-derived Stem Cells with Fascia Fibroblasts
|School||Guangzhou Medical College|
|Course||Obstetrics and Gynaecology|
|Keywords||Pelvic floor dysfunction disease PFD adipose derived stem cells ADSCs fibroblasts indirect co-culture type I collagen protein|
BackgroundWith the improvement of people’s quality of life and aging of the population, the female pelvic floor dysfunction disease (PFD) increased year by year,it is a common disease in older women. The muscles、fascia、ligaments and nerves form a support system of the female pelvic floor. They support and maintain the uterus、bladder and rectum and other pelvic organs in the normal position.Collagen、ligaments and fascia is the main component of these support structures,they play an important role in the flexibility and toughness.Research show that reduce the collagen of ligaments、fascia and other anatomical structures will reduce tension, then will lead to pelvic floor disorders (PFD). There are 21 kinds of human collagen molecules. Bladder and urethra and pelvic floor fascia ligament is mainly composed of collagen I.Collagen I has high tensile strength, supportive role. Many studies indicate that with ageing, expression of type I collagen of pelvic floor fibroblasts decrease. Pelvic connective tissue fibroblasts decrease, I collagen decrease eventually lead to the PFD【1】.Applicating the patch to repair the pelvic floor defects is the main treatment, including the commonly used patches of synthetic patch materials and biological materials,many domestic and international study confirmed synthetic patch with more complications: erosion rate and corrosion rate is about 13% -26%【2】【3】, bio-patch, while having good biocompatibility, but after the implantation of human,degradation has been the clinical focus after 6 months is up to 40%【4】. Therefore, we will search for new materials for the treatment of pelvic floor disorders.Stem cells can self-renew and differentiate. The main characteristics is that stem cells can proliferate and split. 2001, Zuk【5】first get adipose derived stem cells, ADSCs from human adipose tissue. Adipose-derived stem cells has the advantage of: 1、A rich source of material is to facilitate from the human body without injury; 2、Stem cell abundance, a small amount of fat tissue can get many ADSCs; 3、Easier to train; 4、Proliferative capacity is strong, ADSCs is the fastest growing stem cells. Therefore the use of ADSCs has good prospects.Objective:Acording to Unlimited division and proliferation and multi-differentiation potential of adipose derived stem cells,we isolate and subculture and identify ADSCs,then coculture with ligament fibroblasts.At last we will account the abilitity of expressing collagen type I of ADSCs. We will find the new cell to express collagen.Find seed cells for the treatment of pelvic floor dysfunction disease.Materials and Methods1、Adult SD rats of either gender, weighing about 220-250g, side adipose tissue from rat inguinal, then get into the decades of dissociation and subculture. Primary cells were obtained by 1 x 104/ml,1x106/ml,1x108/ml cell concentration then inoculated, in 1-7 days with the MTT assay. Identify the 4th passage cell surface markers CD105、CD106、CD44、CD49d.Use HE staining morphology. Use MTT method to get growth curve of 1-13th cells.2、Use the cell which was identified as fat-derived stem cells, while take fibroblasts from fascia.then adipose-derived stem cells and fibroblasts in fascia will co-culture after different time, detecte the expression of I type collagen.Results:1、Form large whirlpool or long spindle cell colonies, cell surface markers CD44、CD106、CD105、CD49d positive rate is 99.59%、0.85%、95.00%、89.56%. 2、MTT growth curves suggest that, after many passages they keep strong cell proliferation. 1、3、5、7 generation in vitro cultured, incubation period is in the 1-2 day, the logarithmic growth phase is in 3 day, the plateau phase is in 4 day, cell growth curve "S "shape. 9-13 generation of the adipose-derived stem cells, incubation period is in the 1-4 day, the logarithmic growth phase is in 5-6 day, the plateau phase is in 7 day, cell growth curve "S "shape.3、The group one is 1 x 104/ml primary cell vaccination, the growth curve shows i the incubation period is in 1-3 days, 3-4 days into the period of slow growth, the 4-6 days in logarithmic growth phase, 7 days into the plateau. 1 x 106/ml primary cell vaccination is group 2, the growth curve shows the incubation period is in 1-2 days, 2-3 days for the slow growth period, the 3-5 days into the logarithmic growth phase, 6 days into the plateau. 1 x 108/ml primary cells inoculated is group 3, the growth curve shows 2-3 days for the slow growth period, 3-4 days into the logarithmic phase, 5 days into the plateau, the cell growth curve is "S" shape. Therefore, the cell of 1 x 106/ml inoculated go into logarithmic phase early, and the logarithmic phase lasts the longest.4、HE staining shows cell morphology: the 4th generation of ADSCs, HE staining has visible round purple nuclei, coarse granular nuclear chromatin, light red cytoplasm. Elongated spindle cells or angular cells.5、Co-cultured for 3 days, 5 days, 7 days experimental group,type I collagen content compareing with the control group is significantly increased (P <0.05), Co-cultured 5 days or 7 days compareing with 3 days of the experimental group express more type I collagen content (P <0.05), the experimental group of Co-cultured for 7 days compareing with 5 days express type I collagen no significantly difference ( P> 0.05).Conclusion1、successfully isolate and culture rat adipose derived stem cells, the surface markers CD44、CD105、CD49d are positive. CD106 is negative.2、the cells continued to 13 generations still be able to maintain strong proliferative capacity and features.3、within a certain range, different inoculum affect the adipose-derived stem cells proliferate. The 1 x 106/ml group inoculated cells get into logarithmic phase early, and the logarithmic phase lasts the longest. 4.、Rat adipose-derived stem cells co-cultured indirect with fascia fibroblasts express more type I collagen.