Collagen mRNA expression in fibroblasts cultured in vitro and fibroblast response to epidermal growth factor stimulation / 中国组织工程研究

ABSTRACT

BACKGROUND: As the seed cells for construction of tissue engineered skin, fibroblasts directly decide the quality of tissue-engineered skin. During in vitro culture, 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.surgically excised prepuce by trypsin and type Ⅰ collagenase digestion. After cultured till 80% confluence, the cells were digested with mixed digescontrast microscope was used for dynamic observation of the cell morphology and growth status, and transmission electron microscopy and anti-vigen gene expression Reverse transcriptase PCR (RT-PCR) was performed for amplification of type Ⅰ and Ⅲ collagen cDNA derived from the total sis of fibroblast response to EGF stimulation The fibroblasts of P10 and P60passage 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.lasts of different passages to EGF stimulation.decreased with cell passaging and 3H-TdR incorporation was lower in P60cells 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).EGF stimulation is weaker than P10 cells, moreover additional EGF in the condition medium has no obvious regulation on the proliferation of P60cell growth, but extremely remarkable on P10 cells, implying along with the increase of cell passage, tritium-thymidine incorporation reduced and regulative capability of EGF on aging fibroblastic growth was also attenuated.