摘要
目的观察胎牛血清和大鼠血清对体外构建大鼠软骨组织的效应,为组织工程软骨筛选出合适的培养方法。方法取大鼠原代软骨细胞,离心后,培养箱中放置48 h,细胞团块会自发形成软骨小结。分为胎牛血清组和大鼠血清组。每隔一天换一次液,连续培养2周。取出软骨组织,称量重量,固定于4%多聚甲醛,经过脱水、石蜡包埋、切片后,经HE(苏木素-伊红)染色,甲苯胺蓝和藏红-固绿染色及细胞免疫荧光检测Ⅱ型胶原(Col2a1)、I型胶原(Col1a1)的表达。结果胎牛血清组的软骨组织重量比大鼠血清组的软骨组织明显增加,差异具有统计学意义(P<0.05)。HE染色结果表明:大鼠血清组的组织形态呈纤维样结构,而胎牛血清组组织形态呈典型的软骨结构。胎牛血清组的组织甲苯胺蓝和藏红染色明显,而大鼠血清组的组织甲苯胺蓝淡染,固绿深染,提示软骨组织退变或非软骨组织。进一步免疫荧光检测软骨标志基因Ⅱ型胶原和Ⅰ型胶原,胎牛血清组Ⅱ型胶原高表达,而大鼠血清组低表达,Ⅰ型胶原在两组的表达和Ⅱ型的表达呈相反的结果,即在大鼠血清组高表达,在胎牛血清组低表达。结论胎牛血清可促进体外软骨组织的形成,而大鼠血清抑制了软骨组织形成。胎牛血清适合体外构建大鼠软骨组织。
Objective To investigate the effects of fetal bovine serum and rat serum on cartilage tissue engineering in vitro,and provide the proper culture method for tissue-engineered cartilage. Methods The rat primary chondrocytes were collected and cultured in incubator for 48 hours after centrifugation,and the cell pellets spontaneously formed the cartilage nodules. The cartilage nodules were divided into the fetal bovine serum（ FBS） group and rat serum（ RS） group. The medium was changed every other day and the cartilage nodules were cultured for continuous 14 days. Then the cartilage tissues were weighed and fixed in 4% paraformaldehyde. The specimens were dehydrated,embedded in paraffin,cut into slices and then stained with hematoxylin-eosin（ HE）,toluidine blue and safranin O-fast green. The expression levels of type Ⅱ collagen（ Col2a1） and type I collagen（ Col1a1） were detected by immunofluorescence. Results The weight of cartilage tissue in the FBS group was much heavier than that in the RS group,with statistically significant difference（ P〈0.05）. The results of HE staining showed that the tissues exhibited fibrous structures in the RS group but typical cartilage structures in the FBS group. The tissues in the FBS group were obviously stained by toluidine blue and safranin O,while those in the RS group were slightly stained by toluidine blue and strongly stained by fast green,which indicated the cartilage degeneration or non-cartilage tissues. The expression levels of type Ⅱ collagen and type I collagen were detected by immunofluorescence. The type Ⅱ collagen was highly expressed in the FBS group and showed low expression in the RS group. The expression level of type I collagen in the two groups was opposite to that of typeⅡ collagen,which was high in the RS group and low in the FBS group. Conclusion Fetal bovine serum can promote the formation of cartilage tissue,while rat serum can inhibit the formation of cartilage tissue. Fetal bovine serum is more suitable for cartilage tissue
作者
贺自克
丁道芳
王上增
薛艳
郑昱新
曹月龙
HE Zike;DING Daofang;WANG Shangzeng;XUE Yan;ZHENG Yuxin;CAO Yuelong(Department of Join, Henan Provincial Hospital of Chinese Medicine, Zhengzhou 450002, China;Shi＇ s Medical Center of Traumatology,Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China;Institute of Orthopedics and Traumatology, Shanghai Academy of Traditional Chinese Medicine, Shanghai 201203, China)
出处
《上海中医药杂志》
2018年第4期86-90,共5页
Shanghai Journal of Traditional Chinese Medicine
基金
国家自然科学基金资助项目(81503598
81373665)
关键词
胎牛血清
大鼠血清
软骨组织
染色
Ⅰ型胶原
Ⅱ型胶原
fetal bovine serum
rat serum
cartilage tissue
staining
type I collagen
type II collagen
