老年小鼠踝关节软骨细胞体外分离及培养鉴定

In vitro isolation,culture and identification of chondrocytes from the ankle joint of aged mice

背景:老年小鼠的关节软骨本身存在一定的磨损,提取难度较大;尤其是踝关节等难以分离关节软骨面的小关节软骨细胞的提取方法更加少见。目的:探究实验室改良两步酶消化法提取老年小鼠踝关节软骨细胞的效果,体外培养观察并评价其生物学特性。方法:选取46周龄老年BALB/c雄性小鼠踝关节,通过实验室改良的0.25%胰酶EDTA、2 g/LⅡ型胶原酶两步酶消化法顺序进行分离提取及体外培养。使用倒置显微镜观察细胞生长形态,锥虫蓝染色并计数原代贴壁细胞,甲苯胺蓝和Ⅱ型胶原免疫荧光染色,二甲基亚甲基蓝显色法测定原代贴壁细胞培养液和消化液中硫酸糖胺多糖水平,CCK-8试剂盒检测软骨细胞增殖进行软骨细胞生物学特性鉴定及评价。结果与结论:①关节软骨经两步酶消化后,细胞全部解离释放,老年小鼠每个踝关节平均收获9×10^(5)个原代贴壁细胞;原代及第1代、第2代细胞贴壁形态为梭形和多角形;②细胞甲苯胺蓝染色可见蓝紫色易染颗粒,Ⅱ型胶原免疫荧光染色可见红色荧光阳性表达;③原代细胞培养液和消化液中硫酸糖胺多糖分别为(42.41±15.00),(65.63±10.00)mg/L;阴性对照中硫酸糖胺多糖分别为(0.35±3.78),(0.21±2.56)mg/L;④原代细胞第6天细胞数量达到最大,第1代和第2代细胞第5天达到最大,同一时间点,第1代细胞的细胞活力大于第2代细胞和原代细胞;⑤结果证实:实验室改良的两步酶消化法可成功分离老年小鼠踝关节软骨细胞,贴壁细胞纯度高、数量多,可成功合成和分泌Ⅱ型胶原及蛋白聚糖,具有良好的生物学特性,其中第1代软骨细胞的活力最高,第2代次之,可用于实验研究。

BACKGROUND:Knee cartilage loss certainly occurs in aged mice,so it is difficult to extract the articular cartilage from the mice.In particular,it is difficult and rarely reported to extract chondrocytes from the small joint on the articular cartilage surface of the ankle joint.OBJECTIVE:To explore the effect of an improved two-step enzyme digestion method to extract chondrocytes from the ankle joint of aged mice,and to observe and evaluate their biological characteristics in vitro.METHODS:The ankle joints of 46-week-old mice were selected,separated and extracted sequentially by the improved two-step enzymatic digestion method in the laboratory with 0.25%pancreatin EDTA and 2g/L type Ⅱ collagenase.Extracted cells were cultured in vitro and cell growth morphology was observed with an inverted microscope.After trypan blue staining,the number of primary adherent cells was calculated.Toluidine blue and type Ⅱ collagen immunofluorescence staining was conducted.Chromogenic method using dimethyl methylene blue was used to determine the content of glycosaminoglycan sulfate in the primary adherent cell culture and digestive fluid.Proliferation of chondrocytes was detected using cell counting kit-8 to identify and evaluate the biological characteristics of chondrocytes.RESULTS AND CONCLUSION:After the two-step enzymatic digestion of the articular cartilage,all cells were dissociated and released.An average of 9×10^(5) primary adherent cells were harvested from each ankle joint of aged mice.Primary cells and the first-and second-generation cells displayed spindle and polygonal morphology.Toluidine blue staining showed blue-purple particles that were easily dyed.Type II collagen immunofluorescence staining revealed the positive expression of red fluorescence.The contents of glycosaminoglycan sulfate in the primary adherent cell culture fluid and digestive fluid were(42.41±15.00)and(65.63±10.00)mg/L,which were(0.35±3.78)and(0.21±2.56)mg/L in the negative control culture fluid and digestive fluid,respectively.The number of primary cells reached the maximum on the 6th day,and the number of the first-and second-generation cells reached the maximum on the 5th day.Meanwhile,the cell viability of the first-generation cells was greater than that of the second-generation cells and the primary cells.To conclude,the improved two-step enzymatic digestion method can successfully isolate chondrocytes from the ankle joint of aged mice.Large amounts of adherent cells with high purity and good biological characteristics can successfully synthesize and secrete type II collagen and proteoglycan.The first-generation chondrocytes have the highest viability,followed by the second-generation cells,which can be used for experimental research.