摘要
背景:成纤维细胞是构建组织工程化皮肤的种子细胞,其质量好坏直接影响组织工程化皮肤的质量。其在体外培养过程中胶原基因表达及对表皮细胞生长因子刺激反应,可以反映不同代次成纤维细胞的增殖能力,为皮肤组织工程筛选合适的种子细胞提供依据。目的:观察体外培养不同代次人成纤维细胞Ⅰ、Ⅲ型胶原基因的表达及对表皮细胞生长因子刺激的反应性,为组织工程化皮肤种子细胞的选择提供部分依据和参数。设计:自身对照实验。单位:潍坊医学院整形外科研究所。材料:实验于2000-09/2002-06在潍坊医学院整形外科研究所完成。实验样本来源于在潍坊医学院附属医院普外科行包皮环切术的6～8岁正常男性儿童(n=20)切除的健康包皮组织,均自愿提供。方法:①成纤维细胞培养:取包皮组织,去除皮下疏松结缔组织,分离真皮和表皮,真皮部分用含100mL/L胎牛血清DMEM培养液终止胰蛋白酶作用,再用200U/mLⅠ型胶原酶37℃条件下消化30min,收集细胞悬液,用血细胞计数板做细胞计数,观察细胞活性,接种于培养皿中,细胞培养达到80%汇合时,用混合消化液消化,镜下见胞质回缩终止消化,以1:2接种入新培养皿,进行传代培养。②细胞鉴定:用相差显微镜动态观察细胞形态变化及生长增殖情况、透射电镜及抗vimentin免疫细胞化学染色。③不同代次成纤维细胞胶原基因表达的分析:抽提不同代次(P0,P5,…,P65)成纤维细胞总核糖核酸,获得样本cDNA,进行逆转录聚合酶链反应,所得反应产物经10g/L琼脂糖凝胶电泳对照分子量标准,分析扩增产物是否为所预期。④氚-胸苷(3H-TdR)掺入量测定对表皮生长因子刺激的反应性:取P10和P60代成纤维细胞,实验组均添加含表皮生长因子的条件培养液,对照组用不加表皮生长因子的条件培养液。细胞培养至80%汇合时,血清饥饿72h。加入含200μL/L表皮生长因子无血清DMEM培养液,培养24h;弃上清,各组均加入含氚-胸苷的培养液37MBq/L,再培养24h,收集细胞;用预冷的100g/L三氯乙酸沉淀DNA,再用0.5mL1mol/LNaOH提取,液闪计数仪上测定每分钟放射性脉冲数。主要观察指标:①不同代次成纤维细胞Ⅰ、Ⅲ型胶原mRNA含量。②不同代次成纤维细胞对表皮细胞生长因子刺激的反应。结果:①不同代次成纤维细胞胶原基因的表达:随细胞代次的增加,Ⅰ,Ⅲ型胶原mRNA含量逐渐降低。②不同代次成纤维细胞对表皮细胞生长因子刺激的反应:氚-胸苷掺入量在P60细胞(实验组:132.5±23.6,对照组:124.9±16.8)均低于在P10细胞中(实验组:512.8±56.4,对照组:306.4±22.5);对于P60细胞,实验组与对照组间差异无显著性意义(P>0.05),对于P10细胞,实验组与对照组间的差异有非常显著性意义(P<0.01)。结论:①随成纤维细胞代龄的增加,其合成Ⅰ型和Ⅲ型胶原的量逐渐降低。②P60细胞对表皮细胞生长因子刺激的反应均低于P10细胞,添加表皮细胞生长因子的条件培养液对P60细胞生长增殖无明显调控作用,对P10细胞有非常显著的促增殖作用。提示:随着细胞代次的增加,氚-胸苷掺入量降低,表皮细胞生长因子对衰老成纤维细胞生长增殖的调控能力减弱。③本实验为建立成纤维细胞衰老模型提供了实验依据,同时也为组织工程学、老年学及肿瘤生物学及发展提供科学依据。
BACKGROUND： As the seed cells for construction of tissue engineered skin, fibroblasts directly decide the quality of tissue-engineered skin. During in vitro euhure, collagen gene expression and response to epidermal growth factor （EGF） stimulation of the fibroblasts in different passages can be indicative of their proliferative capability for use as the seed cells for skin tissue engineering. OBJECTIVE： To observe the expression of type Ⅰ and Ⅲ collagen mRNA in fibroblasts cultured in vitro and fibroblast response to EGF stimulation, and thereby providing reference for the selection of optimal seed cells for tissue engineering. DESIGN： Self-controlled experiment. SETTING： Institute of Plastic Surgery, Weifang Medical College. MATERIALS： This experiment was carried out at the Institute of Plastic Surgery, Weifang Medical College between September 2000 and June 2002. The specimens of normal prepuce tissues excised by circumcision were obtained from 20 healthy boys at the age between 6 and 8 years on a voluntarily basis in the Department of General Surgery, Affiliated Hospital of Weifang Medical College. METHODS： ① Culture of fibroblasts： The fibroblasts were obtained from surgically excised prepuce by trypsin and type I collagenase digestion. After cultured till 80% confluence, the cells were digested with mixed digestion solution and inoculated for passaging. ②Cell characterization： Phasecontrast microscope was used for dynamic observation of the cell morphology and growth status, and transmission electron microscopy and anti-vimentin immunocytoehemical staining were performed.③ Analysis of colla- gen gene expression： Reverse transcriptase PCR （RT-PCR） was performed for amplification of type Ⅰ and Ⅲ collagen eDNA derived from the total RNA from the fibroblasts of different passages （P05 P5... P65）, and the products were analyzed with 10 g/L agarose gelatin eleetrophoresis. ④ Analysis of fibroblast response to EGF stimulation： The fibroblasts of P10 and P60 passage were divided into treatment group with stimulation by the conditioned medium containing EGF and control group with treatment with only the conditioned medium. 3H-TdR incorporation assay was performed for analyzing the growth of the fibroblasts in response to EGF stimulation. MAIN OUTCOME MEASURES：① Content of type Ⅰ and Ⅲ collagen mRNA in the fibroblasts of different passages. ② Response of the fibroblasts of different passages to EGF stimulation. RESULTS： ③ The content of type Ⅰ and Ⅲ collagen mRNA gradually decreased with cell passaging and 3H-TdR incorporation was lower in P60 cells without significant difference between the treatment group and control group （132.5±23.6 vs 124.9±16.8, P 〉 0.05） than in P10 cells with, however, significant difference between the two groups （512.8±56.4 vs 306.4±22.5, P 〈 0.01）. CONCLUSION： ① With the increase of fibroblastie passage, synthesis of type Ⅰ and Ⅲ collagen gradually reduced. ② Response of P60 cell to EGF stimulation is weaker than P60 cells, moreover additional EGF in the condition medium has no obvious regulation on the proliferation of P60 cell growth, but extremely remarkable on P60 ceils, implying along with the increase of cell passage, tritium-thymidine incorporation reduced and regulative capability of EGF on aging fibroblastic growth was also attenuated.
出处
《中国临床康复》
CSCD
北大核心
2005年第34期150-151,i0006,共3页
Chinese Journal of Clinical Rehabilitation
基金
山东省教育厅重点资助项目--复合人工皮的研究(03K07)~~
作者简介
高学军，男，1971年生，山东省平度市人，汉族，1995年潍坊医学院毕业，学士，主治医师，现为天津医科大学2003级硕士研究生，主要从事组织工程，普通外科方向的研究。
通讯作者：蔡霞，潍坊医学院整形外科研究所，山东省潍坊市261042
