应用自体血清培养大鼠关节软骨细胞生物学行为研究  

Cytobiological Behaviour of Articular Chondrocytes Cultured with Autologous Serum in Rats

作　　者：段小宁[1] 张继英[1] 傅欣[1] 林霖[1] 薛涛[2] 于长隆[1] 

机构地区：[1]北京大学第三医院运动医学研究所,北京100191 [2]北京大学首钢医院

出　　处：《中国运动医学杂志》2011年第3期244-249,264,共7页Chinese Journal of Sports Medicine

摘　　要：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 behaviors of rats＇ articular chondrocytes cultured with autologous serum（AS）and fetal bovine serum（FBS）media in vitro.Methods Materials Articular chondrocytes were isolated from 8-week male SD rats and seeded into the medium with 5% autologous serum（AS）,10% autologous serum（AS）,and 10% fetal bovine serum（FBS）,respectively for monolayer subcultivation.Cell morphological changes were observed by light microscope and growth curves were drawn to determine cell growth rates.Biological behaviors of chondrocytes were analyzed through toluidine blue staining,typeⅠ and typeⅡ collagen immunohistochemical staining,flow cytometry for CD26,CD44,and typeⅠ and typeⅡ collagen expression.Results（1）Polygonal shape of chondrocytes of primary culture developed to spindle shape at passage 3 with no exception in the three groups;（2）Chondrocytes growth rate in 5% AS group was similar to that in 10% FBS group,whereas slower than that in 10% AS group;（3）The excretion amount of acid mucopolysaccharide protein decreased in passage 3 in all groups,and was more obvious in 10% FBS group as compared with that in 5% and 10% AS groups,whereas there was no difference between the two AS groups;（4）The expression of type Ⅱcollagen in 5% and 10% AS groups was higher than that in 10% FBS group（P 0.05）with no significant difference between the two AS groups（P 0.05）.The expression of type Ⅱcollagen decreased gradually in the three groups and the expression of type I collagen increased gradually.（5）The expression of CD44 increased during the culture in the three groups with significant differences between primary and passage 3（P 0.05）,while the expression of CD26 showed no significant differences in the three groups under the same cultured conditions.Conclusion This study found that chondrocytes cultured in 10% autologous serum revealed a higher growth rate and better preservation of phenotype,probably indicating that there could be a possibility to util

关 键 词：自体血清 关节软骨细胞 反分化 

分 类 号：R873[医药卫生—运动医学]