人甲壳质酶蛋白40通过PI3K/Akt信号通路调控膝骨性关节炎兔软骨细胞的凋亡  

Mechanism of YKL-40 regulating apoptosis of rabbit osteoarthritis chondrocytes via PI3K/Akt signaling pathway

作　　者：田胜兰[1] 王国延 杨扬[2] Tian Shenglan;Wang Guoyan;Yang Yang(Wuhan University of Science and Technology Hospital,Wuhan 430065,Hubei Province,China;School of Clinical Medicine,Wuhan University of Science and Technology,Wuhan 430064,Hubei Province,China)

机构地区：[1]武汉科技大学医院,湖北省武汉市430065 [2]武汉科技大学临床学院,湖北省武汉市430064

出　　处：《中国组织工程研究》2020年第32期5108-5113,共6页Chinese Journal of Tissue Engineering Research

基　　金：湖北省卫生厅科研项目(WJ2017M178),项目负责人:田胜兰。

摘　　要：背景:由于PI3K/Akt信号通路与骨组织生理代谢之间存在着密切的联系,而人甲壳质酶蛋白40(YKL-40)对乳腺癌发病机制中的PI3K/Akt信号通路具有一定调控作用,由此推测YKL-40可能通过PI3K/Akt信号通路对膝骨性关节炎软骨细胞凋亡进行调控。目的:研究YKL-40通过PI3K/Akt信号通路调控膝骨性关节炎兔软骨细胞凋亡的作用机制。方法:①将新西兰大白兔随机分为2组,膝骨性关节炎模型组采用前交叉韧带离断术制作右后膝骨性关节炎动物模型,正常对照组仅切开右后膝关节囊,造模后第6周取材并分离软骨细胞,予以软骨组织苏木精-伊红染色及Mankin组织学评分,同时免疫组化染色检测软骨细胞Ⅱ型胶原表达;②正常对照组兔第2代软骨细胞为正常对照组;将膝骨性关节炎模型组兔第2代软骨细胞分为4组,模型组仅予以含体积分数10%胎牛血清的高糖DMEM培养基培养,模型+YKL组培养基中加入100μg/L YKL-40干预,模型+LY组培养基中加入50μmol/L PI3K通路抑制剂LY294002干预,模型+YKL+LY组:培养基中加入100μg/L YKL-40和50μmol/L LY294002干预,采用免疫印迹法检测各组软骨细胞Ⅱ型胶原、基质金属蛋白酶13、Akt、p-Akt、P53、Bcl-2蛋白表达水平。结果与结论:①经软骨组织苏木精-伊红染色、Mankin组织学评分及软骨细胞Ⅱ型胶原免疫组化染色证实,膝骨性关节炎动物模型构建和软骨细胞培养均获得成功;②模型组Ⅱ型胶原、Bcl-2、p-Akt蛋白表达明显低于正常对照组(P<0.05),基质金属蛋白酶13、P53蛋白表达明显高于正常对照组(P<0.05);与模型组比较,模型+YKL组上述指标得到明显改善(P<0.05),而模型+LY组上述指标则进一步恶化(P<0.05),模型+YKL+LY组上述指标与模型组比较无显著性差异(P>0.05),但与模型+YKL组和模型+LY组比较有显著性差异(P<0.05);各组Akt蛋白表达水平比较无显著性差异(P>0.05)。提示:YKL-40可通过激活活化PI3K/Akt信BACKGROUND:Due to the close relationship between the phosphatidylinositol 3-kinase(PI3 K)/Akt signaling pathway and the physiological metabolism of bone tissue,human chitinase protein 40(YKL-40)can regulate the PI3 K/Akt signaling pathway related to breast cancer pathogenesis.Therefore,it is speculated that YKL-40 may regulate apoptosis in knee osteoarthritis(KOA)chondrocytes through the PI3 K/Akt signaling pathway.OBJECTIVE:To study the mechanism by which YKL-40 regulates apoptosis in rabbit KOA chondrocyte s through the PI3 K/Akt signaling pathway.METHODS:(1)New Zealand white rabbits were randomized into two groups.An animal model of KOA was made using anterior cruciate ligament dissection in the model group,whereas the right posterior knee joint capsule was cut but not dissected in the control group.Chondrocytes were extracted from the rabbits at 6 weeks after modeling.Hematoxylin-eosin staining and Mankin histological scoring of the cartilage tissue were performed,whereas immunohistochemical staining was used to detect type II collagen expression in chondrocytes.(2)The second-generation chondrocytes in the control group were used as normal control group,and those in the model group were further divided into four groups,followed by culture with high glucose DMEM medium containing 10%fetal bovine serum in KOA model group,100μg/L YKL-40 in KOA+YKL group,50μmol/L LY294002 in KOA+LY group,and 50μmol/L LY294002+100μg/L YKL-40 in KOA+YKL+LY group.The expression levels of collagen type II,matrix metalloproteinase 13,Akt,p-Akt,P53,Bcl-2 proteins in chondrocytes were detected by western blot.RESULTS AND CONCLUSION:Hematoxylin-eosin staining,Mankin histological score,and collagen type II immunohistochemical staining confirmed the successful construction of KOA animal model and successful chondrocyte culture.Compared with the normal control group,the collagen type II,Bcl-2,p-Akt protein expression levels in chondrocytes were significantly reduced in the KOA model group(P<0.05),and matrix metalloproteinase 13 and P53

关 键 词：膝 骨性关节炎 人甲壳质酶蛋白40 磷脂酰肌醇3-激酶 通路 软骨细胞 凋亡 实验 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学]