低强度脉冲超声经整合素-FAK-p38MAPK通路对膝关节脂肪垫共培养下软骨细胞的影响  

Low intensity pulsed ultrasound can help protect chondrocytes co-cultured with the infrapatellar fat pad through the integrin-FAK-p38 MAPK pathway

作　　者：瞿燕萍[1] 程凯[1] 林强[1] 高明霞[1] 夏鹏[1] 任莎莎[1] 张婷婷[1] 李雪萍[1] 

机构地区：[1]南京医科大学附属南京医院(南京市第一医院)康复医学科,南京210006

出　　处：《中华物理医学与康复杂志》2017年第4期241-246,共6页Chinese Journal of Physical Medicine and Rehabilitation

基　　金：国家自然科学基金（81272151）;南京市医学科技发展基金项目（YKK13113）

摘　　要：目的观察低强度脉冲超声（LIPUS）通过整合素．黏着斑激酶（FAK）-p38丝裂原活化蛋白激酶（MAPK）通路对膝关节脂肪垫共培养下软骨细胞的影响。方法24例女性膝脂肪垫及膝关节软骨均由骨科手术室提供，年龄25～35岁，均为膝关节急性外伤手术患者，排除其他膝关节病史。切取脂肪垫行苏木精-伊红染色（HE）；制作脂肪垫培养液（FCM）；于表面完好部分提取正常软骨细胞进行体外培养，按随机数字表法分成对照组、模型组（正常软骨细胞＋FCM）、治疗组（正常软骨细胞＋FCM＋LIPUS干预）和抑制剂组（正常软骨细胞＋FCM＋LIPUS＋整合素抑制剂GRGDSP干预）。采用Ⅱ型胶原（COL2）免疫细胞化学染色法比较并鉴定对照组与模型组的软骨细胞。治疗组和抑制剂组接受LIPUS辐射，LIPUS辐射强度为40mW／cm2，每次辐射20min，每日1次，每周6d；其余各组接受LIPUS假辐射。第6天收集各组细胞，应用免疫印迹（Western blot）技术检测软骨细胞中Ⅱ型胶原、蛋白多糖（Acan）、基质金属蛋白酶（MMP）-13、整合素B1、磷酸化FAK（p-FAK）、磷酸化p38（p-p38）的蛋白含量。结果免疫细胞化学染色显示，模型组软骨细胞表达的COL2免疫反应物显著少于对照组。Western blot检测显示，模型组COL2、Acan蛋白含量[（0．16±0．04）、（0．12±0．02）]较对照组[（0．55±0．03）、（0．62±0．03）]降低，MMP-13、整合素B1、p-FAK和p-p38蛋白含量[（0．49±0．04）、（0．34±0．04）、（0．33±0．05）和（0．51±0．04）]较对照组[（0．07±0．02）、（0．13±0．03）、（0．16±0．04）和（0．10±0．03）]增高，且组间差异均有统计学意义（P〈0．05）。与模型组相比，治疗组COL2、Aean、整合素BI和p-FAK蛋白含量[（0．42±0．07）、（0．50±0．07）、（0．74±0．06）和（0．64±0．07）]增高，而MMP-13、p-p38蛋白含量[（0．3Objective To observe the effect of low intensity pulsed ultrasound （LIPUS） on chondroeytes co-cultured with infrapatellar fat pads. Methods Twenty-four infrapatellar fat pads and cartilages from female knee trauma patients aged between 25 and 35 were cut and stained using hematoxylin-eosin staining. Chondroeytes were isolated from part of the integrated surface of the cartilages to be cultured in vitro. They were then randomly divided into a normal cbondrocyte group （the control group） , a normal chondrocyte＋FCM （fat conditioned medium） group （ the model group） , a normal chondrocyte ＋ FCM ＋ LIPUS group （ the treatment group） , and a normal cbondrocyte＋FCM ＋LIPUS＋GRGDSP （an integrin inhibitor） group （the inhibited group）. The treatment group and inhibited group received LIPUS at 40 mW/cm2 for 20 rain once a day, while the other groups received sham LIPUS treatment. Five days later, the cells were collected and western blotting was used to examine the expression of type Ⅱ collagen （COL2） , aggrecan （Acan） , matrixmetalloproteinase （MMP） -13, integrin β1, phosphorylation-focal adhesion kinase （p-FAK） and phosphorylation p38 （p-p38）. Results Western blotting showed that compared with the control group, the expression of COL2 and Acan was significantly lower in the model group, but that of MMP-13, integrin β1 , p-FAK and p-p38 was significantly higher. As compared with the model group, the expression of COL2, Acan, integrin β1 and p-FAK was significantly higher, and that of MMP-13 and p-p38 was significantly lower in the treatment group. The expression of COL2, Acan, MMP-13, integrin β1, p-FAK and p-p38 showed no significant difference between the inhibited and model groups, but that of COL2, Acan, MMP-13, integrin β1, p-FAK and p-p38 was significantly different between the control and treatment groups. Conclusions LIPUS provides a protective effect on chondrocytes through inhibiting the expression of MMP-13 induced by adipokines and the degradation o

关 键 词：低强度脉冲超声 软骨细胞 脂肪垫 脂肪因子 整合素Β1 

分 类 号：R684.3[医药卫生—骨科学]