【摘要】 背景:肌腱断裂是骨科常见的棘手问题,而肌腱细胞中胶原分泌影响到肌腱断裂的修复过程,结缔组织生长因子影响组织器官的纤维化过程,对细胞胶原的分泌有重要的作用,但其对肌腱细胞胶原分泌的作用却鲜有报道。目的:通过不同质量浓度的结缔组织生长因子刺激体外培养的原代鸡肌腱细胞,观察细胞中Ⅰ、Ⅲ型胶原的表达情况。方法:选取8周来亨鸡6只,麻醉处死后取屈趾深肌腱行肌腱细胞原代培养,行Ⅰ、Ⅲ型胶原免疫荧光鉴定;取4代的原代肌腱细胞,通过不同质量浓度(0,5,10,20,50,100,200,500μg/L)的结缔组织生长因子刺激4 d,分别行实时荧光定量PCR及酶联免疫吸附测定法检测不同组别肌腱细胞中Ⅰ、Ⅲ型胶原mRNA及蛋白的表达情况。结果与结论:(1)Ⅰ、Ⅲ型胶原免疫荧光鉴定原代培养的细胞为肌腱细胞;(2)实时荧光定量PCR检测提示结缔组织生长因子刺激后肌腱细胞中Ⅰ、Ⅲ型胶原mRNA的表达量均增加;(3)酶联免疫吸附测定法检测提示结缔组织生长因子刺激后肌腱细胞中Ⅰ、Ⅲ型胶原蛋白的表达量均增加(P <0.01),且两者均随结缔组织生长因子质量浓度的升高而逐渐增高,20μg/L及50μg/L的结缔组织生长因子可能是体外刺激肌腱细胞分别生成Ⅰ、Ⅲ型胶原的最佳作用浓度;(4)在一定范围内,结缔组织生长因子能够促进鸡原代肌腱细胞中Ⅰ、Ⅲ型胶原的分泌。更多还原

【Abstract】 BACKGROUND: Tendon rupture is a common thorny problem in orthopedics, and collagen secretion in tenocytes affects the repair process of tendon rupture. Connective tissue growth factor affects the fibrosis process of tissues and organs, and plays an important role in the secretion of cell collagen. However, its effects on the secretion of tenocyte collagen have been rarely reported.OBJECTIVE: To investigate the expression of type Ⅰ collagen and type Ⅲ collagen in primary chicken tenocytes stimulated in vitro with different concentrations of connective tissue growth factors. METHODS: Six Leghorn chickens, aged 8 weeks, were killed, and deep digital flexor tendon was obtained to culture primary tenocytes. Immunofluorescence staining for type Ⅰ and Ⅲ collagen was used to detect the primary tenocytes. Then passage 4 tenocytes were stimulated with connective tissue growth factors at different concentrations(0, 5, 10, 20, 50, 100, 200, and 500 μg/L) for 4 days. Real-time fluorescence quantitative PCR and enzyme-linked immunosorbent assay were used to detect the mRNA and protein expression of type Ⅰ collagen and type Ⅲ collagen in different groups of primary tenocytes. RESULTS AND CONCLUSION: The primary cultured cells were identified as tenocytes by type Ⅰ and Ⅲ collagen immunofluorescence staining. Real-time fluorescence quantitative PCR detection showed that the mRNA expression of type Ⅰ collagen and type Ⅲ collagen was increased in tenocytes after connective tissue growth factor stimulation. Enzyme-linked immunosorbent assay detection showed that the protein expression of type Ⅰ collagen and type Ⅲ collagen was increased in tenocytes after connective tissue growth factor stimulation(P < 0.01), and both increased gradually with the increase of connective tissue growth factors concentration. Connective tissue growth factors at 20 and 50 μg/L might be the best concentrations to stimulate primary tenocytes to produce type Ⅰ collagen and type Ⅲ collagen in vitro. Therefore, connective tissue growth factors within a certain concentration range can promote the secretion of type Ⅰ collagen and type Ⅲ collagen in primary chicken tenocytes.更多还原