【作者】 王鹏；

【导师】 李小林；

【作者基本信息】 南昌大学 ， 外科学， 2010， 硕士

【摘要】 目的:随着社会经济的发展和物质生活水平的提高,人们对容貌美的要求越来越高,更加注重荣光焕发的“面子”工程。而早期的面部皱纹及微小缺损等治疗一直是困扰人们的一个难题,也是美容外科领域所面临的一个新挑战。目前,在美容整形项目中,非手术性治疗迅速增长,占据的比例已经超过了80%。其中,皮肤充填剂注射是非手术性美容治疗的一个重要组成项目,也是治疗早期静态性皱纹及面部微小凹陷的首选方法。早期的皮肤充填剂有很多种,大体可以分为以下3类:（1）人工合成材料如:硅胶、聚甲基丙烯酸甲酯、聚丙烯酰胺水凝胶等。（2）生物材料如:胶原、透明质酸等。（3）自体材料如:自体脂肪或自体胶体等。然而,长期的临床实践表明这些材料均或多或少地存在一定程度的并发症,其中一些材料因并发症较多且较严重已陆续被一些国家禁止使用。作为理想的皮肤充填剂必须符合下列特征:①组织相容性好;②无过敏反应;③不致癌,不致畸;④作用持久,效果可靠;⑤获取容易,成本低廉。目前,采用体外培养的自体皮肤成纤维细胞（human skin fibroblast, HSF）注射移植为解决这一难题提供了一个全新的思路。因为这种方法注入的成纤维细胞是自体细胞,所以不存在免疫排斥反应;其次,注入的成纤维细胞可以在体内存活并增殖,不断地产生胶原蛋白及细胞外基质成分,从而维持持久的填充效果。第三、皮肤标本的获取较为容易,一般只需在患者耳后较隐蔽部位取约3mm2大小皮肤,即可在体外进行分离培养。然而,培养的自体细胞在注射之前需要达到一定的浓度才能移植。如何在体外快速扩增自体成纤维细胞至注射所需浓度,是目前这项技术应用于临床的主要瓶颈。生长因子是一类具有诱导和刺激细胞增殖与分化,维持细胞存活等生物学效应的蛋白类物质,对促进细胞增殖,组织或器官修复和再生都具有重要的促进作用。转化生长因子-β1 （transforming growth factor-β1, TGF-β1）被认为是最重要的致纤维化细胞因子,其在创面愈合和组织纤维化过程中起重要的调控作用,表达水平与瘢痕的增生程度成正相关。主要作用为刺激成纤维细胞中葡萄糖与氨基酸的转运和糖酵解的进行,导致合成显著增加,包括胶原蛋白、粘连蛋白及细胞外基质成分。胰岛素样生长因子-Ι（insulin-like growth factor-I, IGF-I）,是一种与胰岛素具有很大同源性的蛋白质激素,能促进成纤维细胞快速进入细胞周期,刺激细胞分化,明显提高细胞外基质合成。本实验选用以往研究证实的在创伤后瘢痕愈合过程中发挥重要作用的细胞因子即转化生长因子-β₁ （TGF-β₁）及胰岛素样生长因子-Ι（IGF-I）,作用于体外培养的皮肤成纤维细胞,观察各生长因子单独应用与联合在一起应用对皮肤成纤维细胞增殖的影响,探索皮肤成纤维细胞体外快速扩增的新方法,以期缩短临床治疗时间和治疗周期,使自体皮肤成纤维细胞注射移植具有更切实际的临床应用价值。方法:1、皮肤成纤维细胞的原代培养采用胰酶消化法和组织块贴壁法相结合的方法进行原代培养。2、皮肤成纤维细胞的传代培养采用差速贴壁法对其进行纯化,并在倒置相差显微镜下观察细胞形态。3、加入生长因子并检测细胞增殖情况取生长状况良好的第3代皮肤成纤维细胞种入96孔培养板,加入含不同浓度TGF-β₁ （ 0.1、1.0、10.0、50.0μg/L）、IGF-I （ 1.0、10.0、50.0、100.0μg/L）的完全培养液作用于细胞,每个浓度设6个复孔和阴性对照组,分别作用3d、6d和9d,采用MTT法检测细胞增殖情况,确定各生长因子最佳效应浓度。同法设4个组,每组6个复孔,分别为:阴性对照组、TGF-β₁最佳效应浓度组、IGF-I最佳效应浓度组、TGF-β₁与IGF-I最佳效应浓度联合应用组,作用3d、6d和9d,采用MTT法检测各组细胞增殖情况。结果:1、原代培养4d,倒置相差显微镜下观察见少量细胞从组织块边缘爬出,聚集成一团,呈圆形或类圆形,细胞器少,为低分化细胞;7d后细胞逐渐增多,呈梭形散在分布,胞浆周围可见2～3个伪足;传至第3代时细胞已呈明显长梭扁平状,胞核比例增大,细胞器较为明显,为高分化状态。2、TGF-β₁对皮肤成纤维细胞增殖的影响作用6d和9d,各浓度实验组与对照组比较差异均有统计学意义（P<0.05）;10.0μg/L的TGF-β₁与其他各组比较差异均有统计学意（P<0.05）,此即为最佳效应浓度。其他各组间比较差异无统计学意义（P>0.05）。3、IGF-I对皮肤成纤维细胞增殖的影响作用6d和9d,各实验组与对照组比较差异均有统计学意义（P<0.05）。作用9d,50.0μg/L的IGF-I与其他各组比较差异有统计学意义（P<0.05）,此即为最佳效应浓度,其他各组间比较差异无统计学意义（P>0.05）。4、TGF-β₁与IGF-I最佳效应浓度联合应用对皮肤成纤维细胞增殖的影响10.0μg/L TGF-β₁与50.0μg/L IGF-I联合作用9d可显著促进细胞增值,与其他组比较差异有统计学意义（P<0.05）;10.0μg/L TGF-β₁与50.0μg/L IGF-I两组间比较,差异无统计学意义（P>0.05）。结论:TGF-β₁、IGF-I单独应用均可促进体外培养的人皮肤成纤维细胞增殖,两者最佳效应浓度联合应用,促增殖效果优于各自单独使用。更多还原

【Abstract】 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:①Good histocompatibility;②No allergic reaction;③Non-carcinogenic, non-teratogenic;④Lasting effect;⑤L ow cost;⑥Easy 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.更多还原