Background: Fibroblasts were the main seed cells in the studies of tissue engineering of the pelvic floor ligament. Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) were widely studied but at various concentrations. This study aimed to optimize the concentrations of combined bFGF and EGF by evaluating their effects on proliferation and collagen secretion of fibroblasts.
Methods: Fibroblasts were differentiated from rat adipose mesenchymal stem cells (ADSCs). Flow cytometry and immunohistochemistry were used for cell identification. The growth factors were applied at concentrations of 0, 1, 10, and 100 ng/ml as three groups: (1) bFGF alone, (2) EGF alone, and (3) bFGF mixed with EGF. Cell proliferation was evaluated by Cell Counting Kit-8 assays. Expression of Type I and III collagen (Col-I and Col-III) mRNAs was evaluated by real-time quantitative reverse transcription-polymerase chain reaction. Statistical analysis was performed with SPSS software and GraphPad Prism using one-way analysis of variance and multiple t-test.
Results: ADSCs were successfully isolated from rat adipose tissue as identified by expression of typical surface markers CD29, CD44, CD90, and CD45 in flow cytometry. Fibroblasts induced from ADSC, compared with ADSCs, were with higher mRNA expression levels of Col I and Col III (F = 1.29, P = 0.0390). bFGF, EGF, and the mixture of bFGF with EGF can enhanced fibroblasts proliferation, and the concentration of 10 ng/ml of the mixture of bFGF with EGF displayed most effectively (all P < 0.05). The expression levels of Col-I and Col-III mRNAs in fibroblasts displayed significant increases in the 10 ng/ml bFGF combined with EGF group (all P < 0.05).
Conclusions: The optimal concentration of both bFGF and EGF to promote cell proliferation and collagen expression in fibroblasts was 10 ng/ml at which fibroblasts grew faster and secreted more Type I and III collagens into the extracellular matrix, which might contribute to the stability of the pelvic floor microenvironment.
不同浓度碱性成纤维细胞生长因子和表皮生长因子对促进成纤维细胞增殖和胶原蛋白表达的作用与盆底组织再生相关性研究摘要背景：在盆底韧带组织工程的研究中，成纤维细胞是主要的种子细胞。碱性成纤维细胞生长因子（bFGF）和表皮生长因子（EGF）研究广泛、但浓度多样。本研究旨在优化bFGF和EGF的作用浓度，并探索其对成纤维细胞的增殖和胶原蛋白表达的作用。 方法：提取大鼠脂肪进行脂肪间充质干细胞原代培养，并将其诱导分化为成纤维细胞，采用流式细胞术和免疫组化方法进行细胞表型鉴定。生长因子被分为三组：1）bFGF单作用组，2）EGF单作用组，3）bFGF+ EGF联合作用组，浓度均分为0,1,10,和100 ng/ml四种进行作用。以CCK8检测法检测细胞增殖，通过RT-PCP技术检测成纤维细胞I型和III型胶原蛋白mRNA的表达水平。数据分析采用SPSS软件和GraphPad Prism软件进行单因素方差分析和多样本t检验。 结果：脂肪间充质干细胞提取成功并进行原代培养，并完成CD29, CD44, CD90和CD45的细胞表型流式鉴定，成纤维细胞诱导分化成功，且伴随I型和III型胶原蛋白表达增高(F=1.29,P=0.0390)和FSP-1的表达阳性。bFGF组、EGF组和bFGF+ EGF组中浓度为10ng/ml的实验组中促进成纤维细胞增殖作用最强(all P<0.05)，10ng/mlbFGF联合 EGF作用组中的I型和III型胶原蛋白的mRNA表达水平最高(all P<0.05)。 结论：bFGF和EGF的优化作用浓度为10ng/ml，该浓度作用下成纤维细胞增殖更快，I型和III型胶原蛋白分泌更多，有助于在体外重建组织工程中维持盆底组织细胞微环境的稳定性。.
Keywords: Adipose Mesenchymal Stem Cells; Basic Fibroblast Growth Factor; Epidermal Growth Factor; Fibroblasts; Tissue Engineering.