The Infulence Of Antisense Nucleotide Of BMP-2 To The Growth Activeness Of Fibroblast In Skin Scar Tissues And The Collagen Synthesis

Background: The scar is still a difficult problem that hasn't been solved in the scientific field at present. With the improvement of the cure rate of burn , the scar after the deep burn injured has become the main difficult problem in the burn and plastic surgery. It is concerned question all the time that how to make the burn cicatrize at early time and redude or avoild the scar (carry-over). The basic pathology change of scar is that the extracellular matrix deposites. It is maily because of the disorderly deposition and unblanced proportion of collogan. Fibroblast is the key functional cell for the collagen synthesis and the secretion. Its growth mutiplication and the collagen synthesis are closely related to the scar.The wound of embryo can heal without scar and this discovery prompts us that it is possible for the wounds of our humanity to be healed with non-scar. A scholar have found that it is related with bone morphogenetic protein (BMPs).BPMs is the ultra family member of transforming growth factor -β(TGF-β). At first, it is seperated from the bone tissue by Urist. It is taken seriously due to its role of inducing bone. With the research deeper and deeper, it is discovered that BMPs is a multi-function factor and it plays an important part in aspects of embryogeny and growth, the differentiation and multiplication of tissues and cells and so on. In the animal growth, BMPs and the corresponding acceptor are nearly spreading in all internal organs and the body surface organ of the animal.The research on the relations between BMPs and skin mainly focuses on the epidermal cell. Many kinds of bone shape proteins such as BMP-2, BMP-4, BMP-6 and so on have been reported. BMP-2 is one of most active cell factors in BMPs and it has close relations to fibroblast, so former studies concentrate in the fibroblast around the bone and joints, but now there are no deep research report on skin and scar fibroblast.The researches of Stelnicki EJ and so on indicates that BMP-2 expresses low level in the embryo skin tissues, but when adding the exogenetic BMP2 and in its function, the embryo injured area leaves a scar like the adult heals, which means that the BMP-2 participate in the scar's formation. In experiment, Stelnicki EJ discovered that BMP-2 had expressions in the epidermal cell and fibroblast. What's the expression of BMP-2 in adult scar tissues? Any distinguishes of the BMP-2's expression in fibroblast of the human scar and in the normal skin? Does it affact to the multiplication growth and collagen's division of the major function cell forming the scar- fibroblast? There is no related report at home and abroad at present, therefore, we design this experiment, examine the expression of BMP-2 in the normal skin tissues and in the fibroblast through immunlhistochemistry, RT-PCR and other methods, observe its influence to fibroblast's function to confirm BMP-2's function mechanism in the formation of scar through antisense nucleotide disturbing BMP-2's expression.Chapter 1 The BMP-2 expression in the tissues of skin,scar and keloidObjective: To study BMP-2 expression in the tissues of normal skin, hypertrophic scar and keloid and to discuss its relations with the formation of the scar.Methods: Collect 54ps of specimen, among which the normal skin, hypertrophic scar and keloid are for each 18. Slice, the immunlhistochemistry dyeing, observes the BMP-2 expression in tissue blocks, then statistics and analysis.Results: 1. BMP-2 has expressions in the tissues of the normal skin, hypertrophic scar and keloid. It also has expressions in the epidermal cell and the fibroblast.2. BMP-2 expressions in the hypertrophic scar and keloid are much more obvious than that in normal skin and the distiguishes are of statistics significance. But the distiguishes in the hypertrophic scar and keloid are of non-statistics significance.Conclusion: The expression of BMP-2 in hypertrophic scar and keloid is higher than that in normal skin. Chapter 2 The BMP-2 expression situation in the skin andscar fibroblastObjective: To study BMP-2 expression in the normal skin and the scar fibroblastMethods: 1,Cell culture: In the aseptic condition, take 3ps of hypertrophic scar and 3ps of skin raise fibroblasts with the tissue block to paste the wall, then transfer the generation 2,Using RT-PCR to examine the mRNA expression of BMP-2 in the skin and hypertrophic scar fibroblasts. 3,Using Western blot to examine protein expression of BMP-2 in the skin and hypertrophic scar fibroblasts.Results: 1,The Western blot's result shows that the BMP-2 expression is very few in the normal skin tissues, but the protein expression of BMP-2 in the scar tissues increases obviously. There is remarkble difference in statistics (P<0.05).2,The result of RT- PCR shows that mRNA of BMP-2 expression is very few in the normal skin tissues, but the mRNA expression of BMP-2 in the scar tissues increases obviously. There is remarkble difference in statistics (P<0.05).Conclusion: The mRNA expression and protein expression of BMP-2 increase more obviously in hypertrophic scar that than in the normal skinChapter 3 The infulence of antisense nucleotide to the growth activeness of fibroblast in skin scar tissues and thecollagen synthesisObjective: To study the influence of BMP-2 to the growth multiplication of fibroblast and the function of collagen synthesis and discuss the it function mechanism in scar's forming progress.Methods: 1. Grouping: Divide the fibroblast into 4 groups: groupA, cell + nutrient fluid; group B, cell + nutrient fluid + sense oligonucleotide (SODN); group C, cell + nutrient fluid + mismatch sense oligonucleotides (MODN); group D, cell + nutrient fluid + antisense nucleotide(ASODN). 2. Cell transfection: Conduct the extention dyes of antisense nucleotide and others to scar fibroblast according to the operating manual of dyes 3. Determine the BMP-2 expression situation of each group: Using Western blot to examine BMP-2 expression of each groupAExamine the growth suppression rate: Using the MTT analytic method to determine the growth suppression rate of fibroblast in each group.5.Examine the influence of BMP-2 to fibroblast's function: Using hydroxyproline (hydroxypline, HP) color method to determine the collagen synthesis situation of the cells in four groups; Using RT-PCR to determine the mRNA expression situation if I collagen in in four groups of cells.Results: 1. Antisense nucleotide may suppress the BMP-2 expression: Western blot result shows that the sense oligonucleotide (sODN) and mismatch sense oligonucleotides (mODN) have no obviously inluence, but antisense nucleotide (asODN1, asODN2) can suppress the BMP-2 expression in different degrees, especially, the suppressing rate of asODN2 to the BMP-2 expression reaches 80%.2. The BMP-2 antisense nucleotide suppresses fibroblast's multiplication: The MTT analysis demonstrates that it has non-significance difference on cell growth rate in BMP-2 thesense oligonucleotide and the BMP-2 mismatch sense oligonucleotidesgroup comparing with that in the comparing group in different time, but the BMP-2 antisense nucleotide (asODN2) can suppress the growth and multiplication of fibroblast (P< 0.05).3.BMP-2 antisense nucleotide suppresses the collagen synthesis function of fibroblast: After using the hydroxyproline (hydroxypline, HP) color method analysis, its result shows that BMP-2 antisense nucleotide suppresses the collagen synthesis of cells after decreasing the BMP-2 expression of fibrolast, but the collagen synthesis of cells have no obvious changes in the BMP-2 sense oligonucleotide, the BMP-2 mismatch sense oligonucleotidesgroup and comparing group; The BMP-2 antisense nucleotide can suppress I collagen synthesis of cells, but I collagen synthesis of cells in the BMP-2sense oligonucleotide and the BMP-2 mismatch sense oligonucleotidesgroup have no obvious changes comparing with that in the comparing group.Conclusion: BMP-2 promotes the multiplication and growth of fibroblast. BMP-2 promotes the collagen synthesis of fibroblast.