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目的:比较自体血清与胎牛血清体外培养大鼠关节软骨细胞生物学行为的差异。方法:分离培养雄性8周Sprague-Dawley(SD)大鼠软骨细胞,分别在5%、10%自体血清和10%胎牛血清中进行单层传代培养,采用光镜观察细胞形态变化,绘制生长曲线评估细胞增殖速度,通过甲苯胺蓝染色,Ⅰ、Ⅱ型胶原免疫组织化学染色以及流式细胞仪分析细胞CD26、CD44及Ⅰ、Ⅱ型胶原表达以检测软骨细胞生物学行为的变化。结果:(1)在培养3代内,原代培养的软骨细胞形态呈多角形,而3代培养的细胞都为梭形,三组无明显差别;(2)5%自体血清培养的细胞的生长速度与10%胎牛血清培养的细胞接近,10%自体血清培养的软骨细胞的生长速度快于前两组;(3)甲苯胺蓝染色结果证明三组细胞均随传代数增加酸性粘多糖蛋白分泌量下降,且10%胎牛血清组比10%和5%自体血清培养组下降更明显,而10%与5%自体血清培养组之间无明显差异;(4)免疫组化染色和流式细胞仪检测结果表明10%与5%自体血清培养的软骨细胞Ⅱ型胶原表达高于10%胎牛血清培养的细胞(P<0.05),10%与5%自体血清组无显著性差异(P>0.05);随体外培养时间延长,三组细胞合成的Ⅱ型胶原均逐渐减少,Ⅰ型胶原表达逐渐增多。(5)流式细胞仪观察显示三组CD26表达虽有增加但无显著性差异,CD44表达均随传代数增加而逐渐增加,且在1、3代细胞之间具有显著性差异(P<0.05)。在3种培养条件下,相同代数的软骨细胞CD26和CD44表达无显著性差异。结论:本研究发现10%自体血清培养大鼠软骨细胞生长速度快,且仍可以较好保持细胞表型,推测在可能条件下使用浓度较高的自体血清可以缩短体外培养时间而达到较多细胞保持表型的目的。
Objective: To compare the biological behavior of rat articular chondrocytes cultured with autologous serum and fetal calf serum in vitro. Methods: Chondrocytes were isolated and cultured in male Sprague-Dawley (SD) rats for 8 weeks. The cells were subcultured in 5%, 10% autologous serum and 10% fetal calf serum respectively. Morphological changes were observed under light microscope. Curvilinear method was used to evaluate the proliferation of chondrocytes. Toluidine blue staining, type I and type II collagen immunohistochemistry staining and flow cytometry were used to analyze the expression of CD26, CD44 and type I and type II collagen in chondrocytes to detect the change of chondrocytes biological behavior. Results: (1) In primary cultured chondrocytes, the primary cultured chondrocytes showed a polygonal shape in the third passage, while the third passage was spindle-shaped with no significant difference among the three groups. (2) The cells cultured in 5% autologous serum The growth rate was similar to that cultured in 10% fetal bovine serum, and the growth rate of chondrocytes cultured in 10% autologous serum was faster than that in the first two groups. (3) The result of toluidine blue staining showed that all three groups increased the number of acidic mucopolysaccharides Protein secretion decreased, and 10% fetal bovine serum group than 10% and 5% autologous serum culture group decreased more significantly, while 10% and 5% autologous serum culture group no significant difference; (4) immunohistochemical staining and Flow cytometry results showed that the expression of type Ⅱ collagen in chondrocytes cultured in 10% and 5% autologous serum was higher than that in 10% fetal bovine serum (P <0.05), and no significant difference was found between 10% and 5% autologous serum (P> 0.05). With the prolongation of in vitro culture time, the type Ⅱ collagen synthesized by the three groups of cells decreased gradually and the type Ⅰ collagen gradually increased. (5) Flow cytometry showed that the expression of CD26 in three groups increased, but no significant difference. The expression of CD44 increased gradually with the increase of passage number, and there was significant difference between the 1st and 3rd passage cells (P <0.05 ). There was no significant difference in expression of CD26 and CD44 between chondrocytes of the same algebra under the three culture conditions. Conclusion: In this study, we found that 10% autologous serum cultured rat chondrocytes grew rapidly, and can still maintain a good cell phenotype, speculated that the possible use of higher concentrations of autologous serum can shorten the incubation time in vitro to reach more cells Maintain the phenotype.