CAJ | 학술논문

目的：应用大鼠血清建立大鼠体外软骨细胞退变模型。方法：取出生24 h SD大鼠关节处软骨，Ⅱ型胶原酶多次消化后获得原代软骨细胞，取原代细胞进行实验。软骨细胞分别用含10％胎牛血清的DMEM （对照组）、含50 ng／mL 白细胞介素（ IL ）－1β＋10％胎牛血清的 DMEM （ IL－1β组）、含2．5％大鼠血清的DMEM（2．5％血清组）及含5．0％大鼠血清的DMEM（5．0％血清组）中培养。培养24 h后，观察细胞形态变化，MTT法检测各组细胞的增殖情况，蛋白质印迹法检测增殖细胞核抗原的表达和Ⅱ型胶原及MMP－13的表达，实时定量PCR检测退变相关基因ADAMTS5、MMP－9、Aggrecan和SOX－9的表达。结果：两血清组和IL－1β组的软骨形态均由原来的多角形变成长梭形，且两血清组和IL－1β组均促进软骨细胞的增殖，下调转录因子SOX－9和上调基质降解酶MMP－13、MMP－9、ADAMTS5的表达。结论：大鼠血清具有促进软骨退变的作用，可用于建立体外软骨退变模型。
목적：응용대서혈청건립대서체외연골세포퇴변모형。방법：취출생24 h SD대서관절처연골，Ⅱ형효원매다차소화후획득원대연골세포，취원대세포진행실험。연골세포분별용함10％태우혈청적DMEM （대조조）、함50 ng／mL 백세포개소（ IL ）－1β＋10％태우혈청적 DMEM （ IL－1β조）、함2．5％대서혈청적DMEM（2．5％혈청조）급함5．0％대서혈청적DMEM（5．0％혈청조）중배양。배양24 h후，관찰세포형태변화，MTT법검측각조세포적증식정황，단백질인적법검측증식세포핵항원적표체화Ⅱ형효원급MMP－13적표체，실시정량PCR검측퇴변상관기인ADAMTS5、MMP－9、Aggrecan화SOX－9적표체。결과：량혈청조화IL－1β조적연골형태균유원래적다각형변성장사형，차량혈청조화IL－1β조균촉진연골세포적증식，하조전록인자SOX－9화상조기질강해매MMP－13、MMP－9、ADAMTS5적표체。결론：대서혈청구유촉진연골퇴변적작용，가용우건입체외연골퇴변모형。
Objective: To establish a model of chondrocyte degeneration in vitro. Methods:Chondrocytes were isolated from articular cartilages of newly born SD rats by digestion with typeⅡ collagenase .The chondrocytes were cultured with H-DMEM medium containing 10%FBS, 50 ng/mL IL-1β+10%FBS, 2.5% rat serum and 5%rat serum, respectively; and the chondrocytes at passage one were used in the experiments . The morphology changes were investigated under phase contrast microscope after chondrocytes were treated with rat serum and IL-1β.Proliferation of chondrocytes was detected by MTT method .The protein expression levels of PCNA , typeⅡ collagen and MMP-13 were examined by Western blotting . The levels of ADAMTS5, MMP-9, Aggrecan and SOX-9 mRNA were detected by real-time PCR. Results:The cell morphology was changed from polygon to spindle in both rat serum groups and IL-1βgroup, and the proliferation of chondrocytes in these groups was much higher than that in control group .The results showed that the expression levels of typeⅡcollagen, Aggrecan and SOX-9 decreased while the expression levels of MMP-13, MMP-9 and ADMATS5 were up-regulated in rat serum and IL-1β-treated groups compared with control group .Conclusion: The results indicate that rat serum can induce chondrocyte degeneration and may be used for osteoarthritis model in vitro.