Effect Of TGF-β₁, IGF-â…  And Their Synergy On The Proliferation Of Human Skin Fibroblast

Objective:With the socio-economic development and material improvement of living standards,people have paid more attention to the beauty of their own, especially for their face. However, the treatment of early facial wrinkles and minor defects has been a problem to perplex people, and also a new challenge in the field of cosmetology. At present, non-surgical treatment has increased rapidly, and its proportion has occupied more than 80% in all items of cosmetic therapy. Among these,injecting facial fillers was an important component in the project of non-surgical cosmetic treatment. It was also the preferred method to treat the early static wrinkles and facial minor defects. In the early, there were many skin fillers, and these fillers could be roughly divided into the following three categories: first, synthetic materials, such as silicone, polymethyl-mehacrylate, polyacryamide hydro-gel; second, biomaterials, such as collagen, hyaluronic acid; third, autologous tissue, such as fat or colloid. But by long-term practice of clinical we came to a conclusion that these fillers all had complications to a certain extent. Because of more adverse reactions, some of these fillers were banned gradually to use in some countries. As ideal facial fillers, it must meet the following characteristics:â‘ Good histocompatibility;â‘¡No allergic reaction;â‘¢Non-carcinogenic, non-teratogenic;â‘£Lasting effect;⑤L ow cost;â‘¥Easy get. So far, the application of injecting human skin fibroblast( HSF) cultured in vitro has provided a firenew idea for this tough problem. Because these cells injected into skin were autogeneic cells,so there was no hypersensitivity. Besides, these fibroblasts could breed in vivo, and continuously produce collagen protein and extracellular matrix, therefore the fill effects was lasting. Finally, it was very easy to get the skin sample. Generally, we got only 3mm2 size of the skin from the hidden parts behind the patient's ear, then it can be isolated and cultured in vitro. However, autogeneic cells cultured in vitro need to meet a certain concentration before injecting. How to proliferate autogeneic fibroblasts expeditiously in vitro was a bottleneck for the application of this technique in clinical. Growth factor was a class of protein which could induce and stimulate the cell proliferation and differentiation, maintain the cell survival and other biological effects in vivo. At the same time,it played an important role in promoting cell proliferation, tissue or organ repair and regeneration. Transforming growth factor-β₁(TGF-β₁) was considered the most important factor which could induce fibrosis. It played an important regulating role in the process of wound healing and fibrosis. The level of expression of TGF-β₁ was positively related to the degree of scar proliferation. Its main role was to stimulate the transport of glucose and amino acids, and to promote the process of glycolysis. As a result, the synthesis increased significantly, including collagen, fibronectin and extracellular matrix components. Insulin-like growth factor-I (IGF-I ) was a protein hormone which had great homology with the insulin. It could promote fibroblast into the cell cycle quickly, and stimulate cell differentiation, significantly increased extracellular matrix synthesis.In this experiment, transforming growth factor-β₁ (TGF-β₁) and insulin-like growth factor-I (IGF-I ) which were confirmed to play a very important role in the process of wound healing, were chosed to affect fibroblasts cultured in vitro. The purpose of this experiment was to observe the effect of TGF-β₁, IGF-I acting on fibroblasts respectively, and their synergy, in order to explore a new method of expeditious amplification of autogeneic fibroblasts in vitro. Accordingly, the time of therapy and cycle of treatment would be shortened significantly in clinical, so that this technology would have a greater value.Methods:1,Primary culture for skin fibroblasts Trypsin digestion method and tissue adherent method were combined for the cultivation of skin fibroblasts.2,Passaged for skin fibroblasts Differential speed adherent method was applied to depurate skin fibroblasts, and morphous of these cells were observed under inverted phase contrast microscope.3,Growth factors were used for the skin fibroblasts The 3rd passage cells which had a good condition were inoculated into 96 well tissue culture plates, and treated with different concentrations of TGF-β₁(0.1,1.0,10.0,50.0μg/L), IGF-I (1.0,10.0,50.0,100.0μg/L), respectively. The combinations of TGF-β₁ and IGF-I were established at their optimal effect concentrations, and the control group was also established for comparison. Then proliferation of each group was detected at 3days, 6days and 9days by the MTT colorimetric method.Results:1,At 4 days, a small number of fibroblasts crept from the tissue edge when observed under the microscope. These cells were round or oval and gathered up into a ball with few organelles and showing poorly differentiated state. After 7 days, the fibroblasts were scattered and showed fusiform shape. Besides, there were several cell pseudopods around the kytoplasm. When the cells were cultured to third generation, their morphous of these cells were platode or long fusiform and the proportion of nuclei was larger than before. In addtion,organelles were also more evident than before and the cells showed well-differentiated state.2,The effect of TGF-β₁ on the proliferation of human skin fibroblast.The proliferating effects of different concentrations of TGF-β₁ on the 3rd passage fibroblasts at 6 days and 9 days were all significantly better in the growth factor groups than in the control group( P<0.05), and 10.0μg/L of TGF-β₁ was the optimal effect concentration.There were not evident contrast among the other groups ( P>0.05).3,The effect of IGF-I on the proliferation of human skin fibroblast.The proliferating effects of different concentrations of IGF-I on the 3rd passage fibroblasts at 6 days and 9 days were all significantly better in the growth factor groups than in the control group ( P<0.05). At 9 days, the proliferating effects of 50.0μg/L IGF-I was significantly better than any other group. So 50.0μg/L of IGF-I was the optimal effect concentration. There were not evident contrast among the other groups( P>0.05).4,The synergy effect of TGF-β₁ and IGF-I on the proliferation of human skin fibroblast.At 9 days, the combination groups of 10.0μg/L TGF-β₁ and 50.0μg/L IGF-I showed a significantly higher proliferating effect than that in the single growth factor group at their optimal effect concentration( P<0.05), and there was not evident contrast between the two groups of 10.0μg/L TGF-β₁ and 50.0μg/L IGF-I ( P>0.05). Conclusion:TGF-β₁ and IGF-I could promote the proliferation of the HSF respectively, and the combinations of TGF-β₁ and IGF-I at their optimal concentrations had better effects of proliferation than the single growth factor.