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Effects of Retinoic Acid in the Osteogenic Induction to Dermal Fibroblasts and Adipose Derived Stem Cells

Author: LiuYuFeng
Tutor: YuanLin
School: Southern Medical University,
Course: Human Anatomy and Embryology
Keywords: fasciology retinoic acid fibroblast adipose derived stem cells osteogenesis
CLC: R681
Type: Master's thesis
Year: 2013
Downloads: 39
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Abstract


BackgroundThe injury and repair of osseous tissue have always been a hot spot these years. And researchers are expecting to find the appropriate seed cell to solve the problem of regeneration of osseous tissue. In2003, professor Yuan Lin raised the theory of fasciology, considered that human body was constructed by two main systems. One is the support and storage system, which consist of connective tissues. The other is the functional system, which consist of differentiated cells that have their own functions. The support and storage system consistently refresh the functional system, at the same time the functional system maintain stability of human body. Stem cell is the nuclei of the support and storage system. The support and storage system supplement the functional system with stem cells that can differentiate into different cells for it to regenerate, as well as provide it a stable environment. The process of the injury tissue’s repairment is actually the stem cells from the support and storage system differentiate directionally into the the injury tissue cells, and to provide nutrition for the injury tissue to metabolize, maintaining the balance of the internal environment. Mesenchymal stem cells are originated from many different tissues. Theirs contributions are consistent with the contributions of connective tissues. What’s more, their differentiation abilities are accorded with the support and storage system. The fibroblast (DFB) from the dermis and the adipose derived stem cells (ADSCs) are both originated from mesoblast mesenchumal stem cells and are similar in biological characteristic and morphology. They both have the ability to differentiate directionally. ADSCs are the stem cells in fascial tissue, they have multi-differentiation ability. DFB are capable of producing fibers and multiple organisms, especially through the process of injury repairmen. The low immunogenicity of ADSCs provides a better environment for allotransplantation, which make ADSCs a luminous point in biological therapy field.There are mainly two cells that participate in the repairmen of injury. They are fibroblast and undifferentiated mesenchymal cells. The process of bone injury repairmen can roughly divided into three steps:the organization of hematoma, the formation of osteotylus and moulding. Scientists found that fibroblast also play an important role in osteogenesis during the process of the organization of hematoma. Thus how to fully use the osteogenesis ability of fibroblast has long been focused on by many researchers. ADSCs were first discovered and separated by Zuk and his fellows. Compared with other kind of adult stem cells, ADSCs have those advantages as follow:a extensive source, easier to obtain, a smaller ravage and without immunological rejection. ADSCs have stronger proliferation and multi-differentiation abilities. They can be induced into adipose cells, cartilage cells, osteocyte, muscle cells, neurocyte and myocardic cells.The traditional osteogenic induced medium (DMEM+AO%FBS+1%bianticollagen+1%glyceric acid sodium phosphate+0.1%dexamethasone+0.5%vitamin C) has been proved leading fibroblast and ADSCs to osteogenesis. However, the mechanism is still unknown. Retinoic acid (RA) is a intermediate product of the metabolization of vitamin A. Some researches suggested that RA plays an important role in the osteogenesis process of mesenchymal cells. Koussoulakou found that RA improved harnpan synizesis and teeth growth of fetal rats. And some domestic studies proved that RA improved calcium nodules sedimentation of fobrablast. Whether the mechanism of the traditional culture medium and RA working on fibroblast’s osteogenesis are the same is of significant meaning on picking mesenchymal cells as seed cell for osseous tissue engineering.ObjectivesCompare the effects of RA and traditional osteogenic induced medium on osteogenesis of DFB and ADSCs. What’s more, to explore the osteogenesis differences between different kinds of mesenchymal stem cells. Thus enrich the basic theory of fascioloy. At the same time provide more supports for osseous tissue engineering on osseous regeneration and repair.1. To separate and culture ADSCs from rats, identify the surface marker and proliferation potential of ADSCs;2. To separate and culture DFB from rats skin and detect the vimentin.3. Using RA and traditional osseous induced medium on those two cells for2weeks above separately, to observe the differences between their osteogenesis. Detecting growing index like the cell growth curve, bone alkaline phosphatase (ALP) activity, alizarin red staining and RT-PCR for the expression of osteocalcin. Analyze the data.Methods1. To draw material from the back of the5to7-day-old SD suckling mice. Then separated DFB through enzyme digestion and cultured. And then test whether they are DFB by morphological and immunohistochemical methods.2. To draw material from the groin of the same mice. Then separated the ADSCs through enzyme digestion and cultured. And then test whether they are ADSCs by morphological and immunohistochemical methods.3. Wait until the3rd generations were growth up, digested them with0.25%pancreatin, then re-suspend the cell with complete medium. Adjust the density to5×104/m and plan them onto the six-hold-plate. Induced both of the two cells through traditional osseous induced medium and RA for2weeks,2ml every hold. Previous researched done by our fellows have found that the most effective concentration of RA on DFB and ADSCs was5-10mol/L. There are6groups in this study:DFB blank control group; DFB traditional group; DFB RA group; ADSCs blank control group; ADSCs tradional group; ADSCs RA group.4. Pick out4time point during this inducement procedure, which were Od,3d,7d,14d, to draw the growth curve of every group. Alizarin red staining and ALP detecting were carried out after two weeks. RT-PCR was done to detected the expression of the osteocalcin.5. We chose SPSS13.0to do the statistics analysis. Use means±standard deviation to indicate the data of the result. Use factor analysis of variance to compare the differences.Result1. The primary DFB were attached within48h. at the beginning, the amount of the cells are small. To change the medium after3d’s culturing. After5-7days, cells were growth in a large amount. They were long fusiform and polygon in morphology, arranged tightly. Detecting the vimentin of the3rd generations and we found that those who were stained in blue was the nucleus of the cells by DAPI, those in green were FITC fluorescence labeling that revealed vimentin.2. The primary ADSCs were attached within48h. at the beginning, the amount of the cells are small. To change the medium after3d’s culturing. After5-7days, cells were growth in a large amount. They were morphologically the same with DFB. We proved them to be ADSCs through identification of the flow cytometry. The result of the surface markers detection were CD29positive, CD106negative and CD49d negative. Osteogenesis and lipogenesis were succeed in vivro.3. We can see from the cells growth curve that the adding of the inducement factors decreased the growth speed of both cells because the growth speed of the blank control group were higher.4. The result of alizarin red staining of both ADSCs and DFB groups showed obvious calcium nodules while nothing in both blank control groups. And the expressions of ALP were of statistical significant (p<0.05).5. The result of RT-PCR showed that osteocalsin was detected in all intervention groups but not detected in the blank control groups.Conclusion1. DFB and ADSCs are both long fusiform and polygon in morphology. They can both form cell colonies. When treated with immunofluorescence, many positive cells were detected in DFB, which suggested that these cells were originated from dermis.2. When detecting the surface markers of ADSCs, the expression of CD29was beyond90%, while CD49was not expressed. CD106was also detected, but weakly positive. Those results suggested that these cells had the characteristic surface markers of stem cells, however, wasn’t originated from hematopoietic system or epidermic system. Those cells have the abilities of osteogenesis and adipogenesis through directional induction, which is consist with mesenchymal stem cells’ multi-differentiation ability. Thus we can deduce that undifferentiated mesenchymal stem cells exist in fascial tissues.3. When culturing with traditional osseous induced medium and RA, calcium nodules were found in both cells with different culturing medium. In addition, the expression of ALP increased compared with each control group (p<0.05). in DFB group, the amount of ALP was more in RA condition, compared with traditional osseous induced medium. However, the result of ADSCs group was on the contrary.4. The results of RT-PCR showed that the expressions of osteocalcin were detected in all intervention groups.In conclusion, both RA and traditional osseous induced medium can improve osteogenesis of DFB and ADSCs, and this ability differs according to different types of cells. The mechanism of it remains to be seen. According to fascialoly, the support and storage system, which takes stem cell as the core, play an important part in human normal physical activities, injury and repairmen. It refresh the functional system through consistently self-regeneration and update. Connective tissue is part of the support and storage system, it contain abundant of fibroblast and adult stem cells. As a result, a complete comprehension of support and storage system must be a novel understanding of tissue injury and repairmen.

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CLC: > Medicine, health > Surgery > Orthopaedic Surgery ( movement system diseases,orthopedic surgery ) > Bone diseases
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