瞿燕萍，程凯，林强，高明霞，夏鹏，任莎莎，张婷婷，李雪萍.低强度脉冲超声经整合素-FAK-p38 MAPK通路对膝关节脂肪垫共培养下软骨细胞的影响[J].中华物理医学与康复杂志,2017,39(4):241-246
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| 低强度脉冲超声经整合素-FAK-p38 MAPK通路对膝关节脂肪垫共培养下软骨细胞的影响 |
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| DOI： |
| 中文关键词: 低强度脉冲超声 软骨细胞 脂肪垫 脂肪因子 整合素β1 |
| 英文关键词: Ultrasound Chondrocytes Infrapatellar fat pads Adipokines Integrin β1 |
| 基金项目:国家自然科学基金(81272151)；南京市医学科技发展基金项目（YKK13113） |
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| 中文摘要: |
| 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β1、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β1、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、Acan、整合素β1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［(0.37±0.06)、(0.37±0.08)］下降，差异有统计学意义(P<0.05)；而抑制剂组COL2、Acan、MMP-13、整合素β1、p-FAK和p-p38蛋白含量［(0.19±0.04)、(0.09±0.03)、(0.51±0.03)、(0.33±0.02)、(0.30±0.05)和(0.53±0.04)］无明显变化，差异无统计学意义(P>0.05)，但与对照组和治疗组相比，差异有统计学意义(P<0.05)。 结论LIPUS可通过整合素β1-FAK-p38 MAPK通路抑制脂肪因子诱导的软骨细胞MMP-13增高，减少COL2、Acan降解，从而对膝关节脂肪垫共培养下的软骨细胞起一定的保护作用。 |
| 英文摘要: |
| Objective To observe the effect of low intensity pulsed ultrasound (LIPUS) on chondrocytes co-cultured with infrapatellar fat pads. MethodsTwenty-four infrapatellar fat pads and cartilages from female knee trauma patients aged between 25 and 35 were cut and stained using hematoxylin-eosin staining. Chondrocytes were isolated from part of the integrated surface of the cartilages to be cultured in vitro. They were then randomly divided into a normal chondrocyte 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 chondrocyte+FCM+LIPUS+GRGDSP (an integrin inhibitor) group (the inhibited group). The treatment group and inhibited group received LIPUS at 40 mW/cm2 for 20 min 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). ResultsWestern 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. ConclusionsLIPUS provides a protective effect on chondrocytes through inhibiting the expression of MMP-13 induced by adipokines and the degradation of COL2 and Acan through activating the integrin-FAK-p38 MAPK pathway. |
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