基于软骨细胞分离培养的骨痹体外细胞模型的建立  

Establishment of Cell Model of Arthralgia Syndrome in Vitro Based on Isolation and Culture of Chondrocytes

作　　者：王欢 唐学章[1] 舒骏[1] 陈玮 王洋[2] 丁海涛[1] 齐蕊涵 杨帆[1] 陈剑[1] 史榕荇[1] WANG Huan;TANG Xuezhang;SHU Jun;CHEN Wei;WANG Yang;DING Haitao;QI Ruihan;YANG Fan;CHEN Jian;SHI Rongxing(China-Japan Friendship Hospital,Beijing 100029,China;Beijing University of Chinese Medicine,Beijing 100029,China)

机构地区：[1]中日友好医院,北京100029 [2]北京中医药大学,北京100029

出　　处：《中国中医基础医学杂志》2023年第3期401-405,共5页JOURNAL OF BASIC CHINESE MEDICINE

基　　金：国家自然科学基金青年项目(81804214)-推拿通过含miR-146a的胞外囊泡调控固有免疫应答维护骨关节炎关节稳态的机制研究;北京市优秀人才培养资助项目(2018000052580G469)-基于lncRNA/miRNA/circRNA调控网络构建探讨推拿维护骨关节炎固有免疫平衡的表观修饰机制;中日友好医院科研基金青年项目(2019-1-QN-3)-炎性肠病治疗中微生物制剂联合用药的选择与相关机制的研究。

摘　　要：目的建立骨痹体外软骨细胞炎症模型,为研究骨关节炎(osteoarthritis,OA)的中医药治疗机制提供细胞学基础。方法采用胶原酶法进行人软骨细胞的分离培养,观察其形态学,Ⅱ型胶原(collagenⅡ,COLⅡ)免疫荧光染色法鉴定软骨细胞,细胞计数试剂(cell counting kit-8,CCK-8)检测各浓度梯度的脂多糖(lipopolysaccharide,LPS)对软骨细胞生长活性的影响,选择浓度分别为500、100、20、10、2 ng·mL^(-1)的LPS经0、4、6、12、24 h诱导软骨细胞后,Elisa法检测各组细胞上清液中白细胞介素6(interleukin-6,IL-6)和基质金属蛋白酶-3(matrix metalloproteinase-3,MMP-3)的含量,筛选最佳干预浓度及时间。结果分离培养的细胞光镜下观察符合软骨细胞形态学,COLⅡ免疫荧光染色呈阳性,具备软骨细胞特征。与对照组比较,加入差异浓度LPS的各实验组软骨细胞存活率(survival rate,SR)随LPS浓度升高呈逐渐下降趋势,而抑制率(inhibition rate,IR)则逐渐上升,呈浓度依赖性。其中高浓度的LPS对软骨细胞生长有一定的抑制作用,与对照组比较差异有统计学意义(P<0.05),其他各组与对照组比较差异均无统计学意义(P>0.05)。软骨细胞上清液中IL-6和MMP-3在LPS浓度为10 ng·mL^(-1),干预时间为24 h时达到最大浓度(P<0.05)。结论本研究成功分离培养软骨细胞,并提供了一种建立骨痹体外软骨细胞炎症反应模型的优选方式,有利于中医药治疗策略在骨痹中的进一步研究。Objective To establish chondrocyte inflammation model of Arthralgia Syndrome in vitro,so as to provide cytological basis for the further development of osteoarthritis(OA)mechanism and traditional Chinese medicine(TCM)treatment.Methods Human chondrocytes were isolated and cultured by collagenase method and their morphology was observed.CollagenⅡ(COLⅡ)immunofluorescence staining was used to identify chondrocytes,and CCK-8 was used to test the effects of LPS of different concentrations on the growth activity of chondrocytes.LPS with the concentrations of 500,100,20,10 and 2 ng·mL^(-1)were selected to induce chondrocytes for 0,4,6,12 and 24 h.The release levels of IL-6 and MMP-3 in cell supernatant of each group were detected by Elisa,and the optimal concentration and time of intervention were screened.Results The isolated and cultured cells were observed under light microscope in accordance with the morphology of chondrocytes,and the immunofluorescence staining of COLⅡwas positive,showing the characteristics of chondrocytes.Compared with the control group,the survival rate(SR)of chondrocytes in the experimental groups was decreased gradually with the increase of LPS concentration,while the inhibition rate(IR)increased gradually in a concentration-dependent manner.High concentration of LPS had a certain inhibitory effect on chondrocyte growth,and the difference was statistically significant compared with the control group(P<0.05),but there was no significant difference in other groups(P>0.05).The maximum release concentration of IL-6 and MMP-3 in chondrocyte supernatant was reached when the LPS concentration was 10 ng·mL^(-1)and the intervention time was 24 h(P<0.05).Conclusion Chondrocytes were successfully isolated and cultured,and an optimal way to establish chondrocyte inflammatory response model in vitro was provided,which is conducive to the further study of TCM in Arthralgia Syndrome.

关 键 词：骨痹 软骨细胞 分离培养 体外 炎症模型 

分 类 号：R274.9[医药卫生—中西医结合]