| Dermal papilla cells play a important role in hair follicle development and hair follicle cycle regulation.The most significant feature of DPCs is to induce hair follicle formation.Dermal papilla cells cultured in vitro have the ability only after the formation of aggregative growth.Study shows that extracellular matrix and growth factor synthesized by dermal papilla cells play an important role in the development proliferation and differentiation of hair follicles and regulation of hair growth cycle,Expression and activity of extracellular matrix of dermal papilla cells is regulated by growth factors secreted by dermal papilla cells.Growth factors are the regulation factors of the specific response of dermal papilla cells.Therefore,studying the relationship between extracellular matrix and growth factor during the aggregative growth.behaviour.of. dermal.papilla. cells.lay.a.experimental. evidence.for.reconstructio-n,development and regeneration of follicle.Dermal papilla cells have. characteristics of aggregative growth When the cells subcultured more than sixth-generation, this biological characteristics will disappear. We have found that when the pH value of the medium is more than 7.6, the low-generation dermal papilla cells would lose their characteristics of aggregative growth. When these cells were cultured in medium whose pH value is 7.2,their aggregative growth characteristics restored.This phenomenon shows that PH value of the medium is closely related to the characteristics of aggregative growth. We can only surmise that pH value of the training environment can impact secretion of growth factors or synthesis of extracellular matrix. The key factors of aggregative growth may exist in these growth factors or extracellular matrix. Therefore,we cultured low-generation dermal papilla cells in mediums with different pH value.to research the relationship among the synthesis of extracellular matrix and secretion of growth factors of dermal papilla cells and aggregative growth in the training environment with different PH value,to find the key factor which can impact aggregative growth behaviour of dermal papilla cells.MethodsSection I: Effects of pH value on aggregative growth pattern of cultured dermal papilla cells1. Using MTT method to detect the proliferation of dermal papilla cells, using ELISA assay to detect the quantity of HGF and bFGF secreted by dermal papilla cells on the third, the sixth, the ninth ,the twelvth,the fifth /fifteenth day.2. Using alcian blue-Schiff staining to detect acid mucopolysaccharide and neutral mucopolysaccharide of dermal papilla cells.3. Using bivariate correlation analysis method to analyze the correlation between OD value measured by MTT and the number of aggregative growth of dermal papilla cells,to analyze the correlation between the quantity of growth factors and the number of aggregative growth of dermal papilla cells, r>0.5,p<0.05, two sets of data have a positive correlation.Section II :The preliminary study of growth factors inducing high generation of dermal papilla cells to form aggregative growth1. Using HGF,bFGF,EGF to induce aggregative growth formation.2. Using alcian blue-Schiff staining to detect acid mucopolysaccharide and neutral mucopolysaccharide of dermal papilla cells3. One-way ANOVA and independent samples t-test are used for comparison among groups by SPSS12.0 analysis software, p<0.05,differences between two sets of data are significant.Section III : The preliminary study on the relationship between HGF and extracellular matrix in aggregative growth of the dermal papilla cells1.Using 4-Methylumbelliferyl-beta-D–xyloside to block the function of proteoglycans synthesis of dermal papilla cells2. Using HGF antibody to neutralize the role of HGF secreted by the low generation dermal papilla cells3. Using ELISA assay to detect the quantity of HGF secreted by dermal papilla cells whose function of proteoglycans is blocked by 4 - Methylumbelliferyl - beta - D– xyloside.4. Using alcian blue-Schiff staining to detect acid mucopolysaccharide and neutral mucopolysaccharide of dermal papilla cells 5. One-way ANOVA is used to analyse these measurement data by SPSS12.0 analysis software, p<0.05,differences between two sets of data are significant.ResultsSection I: Effects of pH value on aggregative growth pattern of cultured dermal papilla cells1. Aggregative growth pattern appeared in the dermal papilla cells cultured in training enveronment whose pH value was 7.2 or pH7.6.2. The alcian blue-Schiff staining showed that dermal papilla cells cultured in training environment whose pH value was 7.2 or pH7.6.synthesized both acid mucopolysaccharide and neutral mucopolysaccharide, which were in the same stain compared with positive control group of low-generation dermal papilla cells, while dermal papilla cells cultured in training environment whose pH value was 6.8 or 8.0 only synthesized neutral mucopolysaccharide,but almost no acid mucopolysaccharide.3. No correlation exists between OD value measured by MTT and the number of aggregative growth of dermal papilla cells, correlation exists between quantity of growth factors and the number of aggregative growth of dermal papilla cellsSection II : The preliminary study of growth factors inducing high generation of dermal papilla cells to form aggregative growth1. Aggregative growth pattern appeared in the eighth-generation dermal papilla cells induced by the 40 ng/mlHGF, but aggregative growth pattern did not occur in other growth factors-induced dermal papilla cells.2. The alcian blue-schiff staining showed that, most cytoplasm of HGF-induced dermal papilla cells appeared both red and blue color. Intensity of red and blue of dermal papilla cells in the aggregative growth region were stronger than those in non- aggregative growth zone, compared with the positive control group,dermal papilla cells alcian blue-Schiff staining is basically the same situation.Section III : The preliminary study on the relationship between HGF and extracellular matrix in aggregative growth of the dermal papilla cells1. No aggregative growth. appeared in the fourth-generation dermal papilla cells co-cultured with 4-Methylumbelliferyl-beta-D–xyloside for two weeks.No aggregative growth pattern occur in the fourth-generation dermal papilla cells co-cultured with 4-Methylumbelliferyl-beta-D–xyloside and 40ng/ml HGF for two weeks. No aggregative growth pattern occur in the fourth-generation dermal papilla cells co-cultured with 40μg/ml HGF antibody for two weeks.2. The alcian blue-Schiff staining showed that the fourth-generation dermal papilla cells co-cultured with 40μg/mlHGF antibody or 4-Methylumbelliferyl-beta-D–xyloside or 4-Methylumbe lliferyl-beta-D–xyloside and 40ng/mlHGF only synthesized neutral mucopolysaccharide, but almost no acid mucopolysaccharide.3. ELISA test results show that the quantity of HGF secreted by dermal papilla cells of the 4-Methylumbelliferyl-beta-D–xyloside group is significantly less than that of the positive control group.(p<0.05).There is no significant difference between the quantities of HGF secreted by dermal papilla cells of 4-Methylumbelliferyl-beta-D–xyloside group and 20μg/mlHGF antibody group at different time points(p>0.05).Conclusions:1. The number of aggregative growth of cultured dermal papilla cells is dependent on the quantity of HGF secreted by dermal papilla cells.The proliferation ability of dermal papilla cells is not correlated.with the number of aggregative growth of cultured dermal papilla cells.2. HGF can induce dermal papilla cells to form the aggregative growth pattern.3. Acidic mucopolysaccharide is the material basis of aggregative growth of dermal papilla cells , HGF has the role in aggregative growth of dermal papilla cells through acid mucopo lysaccharide4. The study suggests that the best PH value interval of training enveronment of dermal papilla cells in vitro is 7.2-7.6.
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