摘要
目的研究丹参酮ⅡA在大鼠膝软骨细胞体外培养中对软骨细胞去分化的影响。方法采用Ⅱ型胶原酶消化法获取2周龄SD大鼠膝关节原代软骨细胞,体外培养传代至P4进行实验。对照组用含10%小牛血清高糖DMEM培养(基础培养液)。实验组有3个浓度:100μg/ml组用基础培养液+100μg/ml丹参酮ⅡA培养,200μg/ml组用基础培养液+200μg/ml丹参酮ⅡA培养,400μg/ml组用基础培养液+400μg/ml丹参酮ⅡA培养。培养72 h后,采用CCK-8法检测各组细胞增值情况,光学显微镜观察软骨细胞形态,阿利新蓝染色观察糖胺聚糖(GAG)含量,实时荧光定量PCR检测Ⅰ型、Ⅱ型、Ⅹ型胶原表达情况。结果 400μg/ml组肉眼可见的细胞密度低于其他各组,且阿利新蓝染色最浅。培养24 h、48 h、72 h,100μg/ml组、200μg/ml组的OD值均大于对照组,而400μg/ml组OD值均小于对照组,差异有统计学意义(P<0.05)。实验组的Ⅰ型、Ⅹ型胶原m RNA含量明显低于对照组,差异有统计学意义(P<0.05)。400μg/ml组Ⅱ型胶原m RNA含量低于对照组,100μg/ml组、200μg/ml组Ⅱ型胶原m RNA含量高于对照组,差异有统计学意义(P<0.05);但100μg/ml组、200μg/ml组差异无统计学意义(P>0.05)。结论不同浓度的丹参酮ⅡA对去分化软骨细胞作用有所区别,100μg/ml、200μg/ml时促进软骨细胞增殖,抑制去分化进程;400μg/ml时抑制软骨生长活性,细胞基质分泌减少。
Objective To investigate the effects of Tanshinon ⅡA on the dedifferentiation of chondroeytes in vitro. Methods The distal femoral cartilage was obtained from the knee joints of two-week-old Sprague-dawley rats. Chondrocytes were isolated from the cartilage with type Ⅱ collagenase digestion method. After cell expansion in vitro, the cells of passage 4 （P4） were used for further study. Four different culture conditions were investigated in this study. Control group： chondrocytes in DMEM medium, 100 μg/ml group： DMEN medium＋100 μg/ml Tanshinon Ⅱ A, 200 μg/ml group： DMEN medium＋200 μg/ ml Tanshinon ⅡA, 400 μg/ml group： DMEN medium＋400 μg/ml Tanshinon Ⅱ A. Seventy-two hours later, the ceils were collected for analysis. Cell Counting Kit-8 （CCK-8） was used to observe cell proliferation. Optical microscope was used to observe the morphology of chondrocytes in the groups. Alcian blue staining was used for observation and comparison of glycosaminoglyean （GAG） content in ehondrocytes. The cartilage-related specific genes including collagen I , H, X were examined by real-time quantitative （RT-qPCR） assay. Results The cell density was observed to be lower in the 400 μg/ml group than that in the other groups, and Alcian Blue staining was also lighter. Compared to the control group, the OD value of 100 μg/m] group and 200 μg/ml group were obviously higher and 400 μg/m] group was lower at 24 h, 48 h, 72 h （P 〈0.05）. The mRNA content of type I and type X collagen in the experimental group was all reduced. Type H collagen of 400 μg/ml group was lowest than other groups（P 〈0.05）, but there was no difference between 100 μg/ml and 200 μg/ml group（P 〉0.05）. Conclusion Different concentrations of Tanshinone Ⅱ A play various roles in the dedifferentiation of chondrocytes. In this study, growth activity and cell matrix secretion was inhibited in 400 μg/ml group while chondrogenesis was suitable in 100 μg/ml and 200 μg/ml group which could inhibit the dedifferentiation of chondrocytes.
作者
张煜珅
张普华
刘鑫成
成西侠
范宏斌
ZHANG Yu-shen ZHANG Pu-hua LIU Xin-cheng CHENG Xi-xia FAN Hong-bin(Department of Orthopedic Surgery, Xijing Hospital, Fourth Military Medical University, Xi＇an, Shan＇xi 710032, China)
出处
《中国骨与关节损伤杂志》
2017年第9期942-945,共4页
Chinese Journal of Bone and Joint Injury
基金
国家自然科学基金(31170936
31470936)
关键词
软骨细胞
去分化
丹参酮ⅡA
组织工程
Chondrocytes
Dedifferentiation
Tanshinon ⅡA
Cartilage
Tissue engineering
