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目的:观察不同血清浓度对小鼠L929成纤维细胞生长增殖和分泌Ⅰ型胶原、Ⅲ型胶原和羟脯氨酸的影响,以了解适宜成纤维细胞生长和分泌的最佳血清浓度。方法:L929细胞分成6组:分别以血清浓度为0 mL/L、200 mL/L、400 mL/L、600 mL/L、800 mL/L和1 000 mL/L的培养液培养,培养2 d、4 d、6 d和8 d分别取各组细胞进行HE和吖啶橙荧光染色,了解细胞形态和生长状况变化。在2 d、4 d、6 d和8 d分别取各组上清,酶联免疫法检测Ⅰ型胶原,Ⅲ型胶原,羟脯氨酸水平。磺基罗丹明B(SRB)法检测培养2 d、4 d、6 d和8 d后各组细胞增殖情况。结果:(1)L929在血清浓度很高和很低(0%和100%)时细胞增殖率都明显下降,20%组血清浓度的增殖率最高;40%~100%组,随着血清浓度增高,细胞增殖率反而下降;(2)HE和AO染色发现0%血清浓度组细胞出现凋亡情况,80%组细胞开始出现空泡化,100%血清浓度时细胞空泡化严重。20%~60%血清浓度细胞形态基本正常。(3)0%、80%和100%血清浓度在培养的8 d内I型胶原浓度一直比较低,40%血清浓度I型胶原浓度最高。培养的第2天,各浓度组的Ⅲ胶原均有增加,后有所下降,6~8 d时又再次上升。各组血清浓度组的L929细胞在不同培养时间羟脯氨酸浓度变化不大。结论:20%~40%血清浓度最适合成纤维细胞的生长,增殖和胶原分泌,血清浓度过高或过低都会引起细胞增殖率下降、细胞凋亡、空泡化和胶原的分泌减少,自体血清注射时采用合适的血清浓度可能会取得更好的效果。
OBJECTIVE: To observe the effects of different serum concentrations on the growth, proliferation and secretion of type I collagen, type III collagen and hydroxyproline in mouse L929 fibroblasts in order to understand the optimal serum concentration for the growth and secretion of appropriate fibroblasts. Methods: L929 cells were divided into 6 groups and cultured in the medium of 0 mL / L, 200 mL / L, 400 mL / L, 600 mL / L, 800 mL / L and 1000 mL / The cells in each group were stained with HE and acridine orange fluorescence staining on day 4, day 6, day 8 respectively to understand the changes of cell morphology and growth status. The supernatant of each group was taken at 2 d, 4 d, 6 d and 8 d respectively. The levels of collagen type Ⅰ, type Ⅲ collagen and hydroxyproline were detected by enzyme-linked immunosorbent assay. Sulfro-rhodamine B (SRB) method was used to detect the proliferation of cells in each group after 2 d, 4 d, 6 d and 8 d. Results: (1) The cell proliferation rate of L929 was significantly decreased at high and low serum concentrations (0% and 100%), while the proliferation rate of 20% serum was the highest in 40% ~ 100% group. With the increase of serum concentration (2) The apoptosis of cells in 0% serum concentration group was found by HE and AO staining. 80% of the cells began to vacuolization, and the cell vacuolization was serious at 100% serum concentration. 20% ~ 60% serum concentration of cell morphology was normal. (3) 0%, 80% and 100% serum concentrations of type I collagen in the 8 d culture has been relatively low, 40% serum concentration of type I collagen highest concentration. On the second day of culture, the collagen Ⅲ in each concentration group increased and then decreased, and rose again at 6-8 days. The concentrations of hydroxyproline in L929 cells of different concentration groups did not change much at different culture time. Conclusion: 20% ~ 40% serum concentration is the most suitable for the growth, proliferation and collagen secretion of fibroblasts. The high or low serum concentration can cause the decrease of cell proliferation rate, decrease of apoptosis, vacuolization and collagen secretion, Serum injection with appropriate serum concentrations may achieve better results.