| Pelvic floor dysfunction (pelvic floor dysfunction, PFD) is a common disease among the old women. Its incidence is about 40 percent.The disease mainly including pelvic organ prolapse (pelvic organ prolapsed, POP) and stress urinary incontinence, (SUI), the two diseases often occurs at the same time,we can guess that they have a same pathogenesis. Many research thought the content of pelvic connective tissue is decreasing in patients with PFD,and they considered that the decreasing of the content of pelvic connective tissue lead to the relaxation of the ligment and thefascia,which eventually brought about the PFD. At present, treatment about PFD basically have conservative treatment and surgical treatment, conservative treatment mainly applies to the lighter patients,while surgical treatment contains the traditional surgery and some new surgical methods.New surgical methods applies biological material to reconstruction the structure of pelvic floor, compared with the traditional surgical,the former one is with less injury, the lower recurrence, bue it exist some problems such as be more susceptible to infection, plant net slice easy to be eroded, be outside leakage, be rejected.Bone marrow mesenchymal stem cells (BMSCs) are from mesoderm, which have very strong self-replicating and multiplex differentiation potential.They can produce offspring cell with various types. In vivo and in vitro,under the different induction conditions, BMSCs can divide into mesoderm and outside nerve mesoderm such as osteoblast, chondrocytes, fat cells, endothelial cells, muscle cells, nerve cells and the stromal cells support hematopoietic etc.Further more, bone marrow MSCs is easily to be obtained relatively, and can obtain from oneself. The condition of BMSCs demand in vitro is not high.The split proliferative capacity of bone marrow MSCs is strong, have various advantages:no allograft rejection and biocompatibility. This discovery for tissue engineering in BMSCs alternative autologous ligament fibroblasts provides theoretical basis for the application.ObjectiveThis research discusses the method of culturing rat bone marrow mesenchymal stem cells (BMSCs) in vitro and its differentiation induction into fibroblasts. It adopts the conditioning culture medium to induct BMSCs to explore the methods to induct rat BMSCs being transformed into ligment fibroblasts. Thus provides basis for the selection of seed cells in construction of ligment tissue engineering.Methodology(1) BMSCs of rat were isolated by using differential adherence method in vitro. They were identified through morphologic method and by phenotype molecule(CD44, CD90, CD45, CD34 for rBMSCs) assessments detected by flow cytometry to testify their differentiation potentiality. And they have been induced to differentiate into osteoblasts and adipocytes.(2) The third generation of BMSCs were commitedly induced for 3days and 6 days by conditioning culture medium (LG-DMEM containing 10%fetal bovine serum,10 ug/L bFGF,1 mmol/L phosphoric acid vitamin C,0.04 mmol/L proline). After they were differentiated into fibroblasts, Growth curve line was used to confirm the proliferation of the induced BMSCs. Immunocytochemical method and Western blot were adapted to detect the expression of typeⅠ,Ⅲcollagen.(3) All data were expressed by mean±standard deviation. In the process of treatment of results in the immunohistochemistry and Western-blot, one-way ANOVA was used to test difference between the sample and the control group. The P<0.05, as the difference between the two groups, is statistically significant.Results(1) BMSCs can be successfully isolated and cultured by means of differential adherence method. The morphologic features were identified to show that the rat BMSCs had the general appearance of stem cells, i. e. larger nuclei and minor plasm. By means of FCM, rBMSCs show the phenotype of positive expression of CD90(98.9%),CD44(98.6%), and negative expression of CD34(1.8%), CD45(3.6%). Moreover, Osteogenic and adipogenic inductions were confirmed by Alizarin and oil red-"0" staining respectively, shows their high differentiation potential successfully.(2) The inducted BMSCs devise actively. A value of the difference between the two groups was significant (P<0.05).(3) The synthesis of collagen was detected by immunocytochemistry and Western-blotdetection. The expressions of typeⅠand typeⅢcollagens were enhanced after being induced for 6 days. The expression of BMSCs collagenⅠ,Ⅲincreased significantly comparing with that of the control group (P<0.05).(4) BMSCs were induced by conditioning culture medium for 3 days. Immunocytochemistry and Western-blot were used to detect the synthesis of collagen. The difference between the BMSCsⅠ,Ⅲcollagen and the control group not statistically significant (P>0.05)Conclusions(1) BMSCs with high differentiation capacity were isolated from rat bone marrow successfully. BMSCs isolated through differentiation adherence method were with high purity and they can be differentiated into bone cells and fat cells.(2) Mesenchymal stem cells were transformed into ligament fibroblasts in a short period through the induction of conditioning culture medium in vitro. By which method the proliferation of BMSCs was enhanced and the synthesis of collagenⅠand collagenⅢwas promoted. |
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