| BackgroundFemale plevic floor disfunction(PFD), as one of the commmon diseases of middle aged and old females, has a main series of symptoms including pelvic organ prolapse, stress urinary incontinence, sexual abnormalities, chronic pelvic pain, abnormal defecation and so on. With the constantly increasing incidence rate, it has become a global public health issue. PFD has not only influenced the life quality, physical and mental health of patients, but also become a heavy financial burden on the community.Pelvic floor support structures include the pelvic floor muscles and pelvic floor connective tissue. Whatever cause the support structures changed also can lead to PFD. Domestic and international studies both found that the number of pelvic floor fibroblasts has decreased and their function has declined in PFD patients, all of these causing a decreasing content of type I and III collagen in connective tissue and a change of pelvic floor support structures. The application of stem cells in the treatment of PFD may be able to replace or repair the lesions or defects of the pelvic floor connective tissue, and reconstruct the anatomy and physiological functions of pelvic floor support structures. Adipose derived stem cells (ADSCs) are one class of mesenchymal stem cells which have proliferation and potential of differentiation. It's advantages include a wide source, drawing easy, minimally trauma, high abundance of stem cells, low immunogenicity and so on. Nowadays, stem cells are mainly used in the treatment of SUI, by injecting stem cells in the araa next to urinary tract to repair damaged urethral sphincter. It has entered clinical trials, but its safety and long-term efficacy are unknown. However, there are little researches about the application of stem cell in the POP.Connective tissue growth factor (CTGF) is a polypeptide growth factors, which can promote a variety of cell to proliferate, differentiate, and secrete extracellular matrix like collagen and so on. However,in so far little is known about the effects of extrogen CTGF on expression of collagen I and collagen III of ADSCs.Part I Isolation, culture, purification and identification of ADSCsObjectiveExplore the method of isolation, culture, purification and identification of ADSCs, and research its basic biological and surface antigens characteristics, its differentiation potential into osteoblasts and adipocytes inorder to provide experimental data for the study of ADSCs.Methods:1. Collagenase was used to digest SD rat inguinal fat pads to isolate ADSCs, and passaging is used to purify ADSCs.2. MTT was used to measure the growth characteristics of P1,P3,P5 ADSCs, the cell cycle and surface markers of P3 ADSCs were detected by flow cytometry, and the purity of P3 ADSCs were analysed at the same time .3. P3 ADSCs was differentiated into osteoblasts by osteogenic medium, alizarin red staining was used to identify its osteogenic.Results: Characteristics of rat ADSCs:1. Morphology: Morphology of primary and passaged rat ADSCs was similar to fibroblasts. It proliferated well in vitro, and P13 ADSCs also retained the good proliferation ability.2. Growth curves: P1,3,5 rat ADSCs had a roughly same growth curve, as "S" shape.3. Cell cycle: 90.6% of P3 rat ADSCs were in the G0/G1 phase, 3.99% in S phase, and 5.41% in G2 / M phase.4. Cell surface markers: The positive expression of cell surface markers of P3 rat ADSCs were listed as followed: CD44 (99.88%), CD49d (89.56%), CD90 (99.74%), CD105 (95%), CD34 (3.04%), CD45 (0.26%), CD106 (0.85%).5. Differentiation potential: Rat ADSCs induced by ostergenic medium formated island-like extracellular deposition, and deposition increased with time. Alizarin red staining was positive after inducted for 14 days.Conclusions:The seperated cells were been proveded to be ADSCs by the characteristics of its cell circle, expressions of cell surface markers and osteogenic differentiation potential. And P3 ADSCs has a high purity of ADSCs. The differentiation potential suggests that ADSCs may become one of the ideal seed cells of pelvic floor tissue engineering.Part II Effects of connective tissue growth factor on expression of collagen I and collagen III of rat adipose tissue-derived stem cellsObjectiveUse CTGF to stimulate rat ADSCs, to survey the effects of extrogen CTGF on expression of collagen I and collagen III of ADSCs and the appropriate conditions between both. To provide heoretical references and methodological references in the use of CTGF and ADSCs in pelvic floor dysfunction treatment and pelvic floor tissue engineering.Methods:1. Experiment of effects between different concentrations of CTGF: Respectively 0,1,10,25,50,100 ng/ml concentration of CTGF had been used to stimulate rat ADSCs for 1,2,3 days in vitro. The expressions of collagen I and collagen III mRNA in ADSCs were been analysed by semi-quantitative RT-PCR. The contents of collagen I and collagen III in cell culture supernatant were been analysed by ELISA.2. Experiment of change of effects of CTGF in a long time culture in vitro: 100ng/ml CTGF had been used to continuous stimulate ADSCs for 7,14,28 days, to observed the changes of reaction in ADSCs in vitro.3. Differentiation potential of osteogenic and adipogenic of the inducted ADSCs by CTGF: ADSCs was stimulated by 100ng/ml CTGF for 14 days, and differentiate the stimulated ADSCs by osteogenic and adipogenic medium, to identify the changes of its differentiation potential of osteogenic and adipogenic. Compared with the control group which was not stimulated by CTGF.4. Statistical analysis: Data analysed through SPSS 16.0. Experimental datas were showed as mean±standard deviation(—x±s)said. P <0.05 indicated significant difference.Results:1. Experiment of effects between different concentrations of CTGF: Collagen I and Collagen III mRNA expressions increased with time in different concentrations of CTGF group, compared with the control group there were significant differences (P <0.05). Collagen I and Collagen III mRNA expressions of 1-100ng/ml CTGF group increased by the role of time-dependent concentration (P <0.05). Collagen I and Collagen III levels in supernatant increase with time in 1-100ng/ml CTGF group, compared with the control group there were significant differences (P <0.05). Collagen I levels of 10ng/ml and 50ng/ml group were lower than 100ng/ml group (P <0.05); however, the difference was not significant between 10ng/ml and 50ng/ml group in the levels of Collagen I (P> 0.05). Collagen III level of 10ng/ml was lower than 50ng/ml and 100ng/ml group (P <0.05); however, the difference was not significant between 50ng/ml and 100ng/ml group in the levels of Collagen III (P> 0.05).2. Experiment of change of effects of CTGF in a long time culture in vitro: Expression of Collagen I mRNA in rat ADSCs at Day7 was 1.069±0.324; Day14 2.162±0.038; and Day28 3.360±0.058. Expression of Collagen III mRNA in rat ADSCs at Day7 was 1.398±0.073; Day14 4.081±0.835; and Day28 4.466±0.049. Expression of Collagen I and Collagen III mRNA were increased with time (P <0.05).3. Differentiation potential of osteogenic and adipogenic of the inducted ADSCs by CTGF:The stimulated cells which cultured in ostergenic medium did not formate extracellular deposition, and alizarin red staining was negative after cultured for 14 days. Lipid droplets were also not formated in the inducted cells cultured by adipogenic medium, and oil red O staining was negative after cultured for 14 days. But the results of control group were opposite.Conclusions:1. The expression and levels of Collagen I and Collagen III mRNA in rat ADSCs increase when by stimulated under CTGF, indicating that CTGF can promote the expression and secretion of Collagen I and Collagen III in ADSCs.2. The expressions of Collagen I and Collagen III mRNA in rat ADSCs is still promoted by 100ng/ml CTGF until has been cultured for 7,14,28 days in vitro,and the expressions increase with time. Collagen I and Collagen III mRNA expression increased with time.3. CTGF has removed the osteogenic and adipogenic differentiation potential of ADSCs, but the mechanism is unkown.. |
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