| 瞿燕萍,高明霞,夏鹏,张婷婷,程凯,林强,李雪萍.膝骨关节炎脂肪垫对关节软骨细胞的影响[J].中国康复,2017,32(3):179-184 |
| 膝骨关节炎脂肪垫对关节软骨细胞的影响 |
| Effect of knee infrapatellar fat pad on articular chondrocytes in osteoarthritis |
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| DOI： |
| 中文关键词: 膝骨关节炎 软骨细胞 髌下脂肪垫 |
| 英文关键词: osteoarthritis chondrocytes infrapatellar fat pad |
| 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：观察人膝骨关节炎(OA)脂肪垫对正常及OA关节软骨细胞的影响。方法：24例人膝脂肪垫和膝关节软骨均由骨科手术室提供，12例为膝关节急性外伤手术患者，12例为膝OA患者行关节置换术后。切取正常及OA膝脂肪垫行苏木精-伊红染色(HE)及瘦素免疫组织化学染色；用OA膝脂肪垫制作脂肪垫培养液(FCM)；于急性膝外伤手术标本的表面完整处提取正常软骨细胞，于膝OA关节置换术标本提取OA软骨细胞，分别进行体外培养并鉴定，随机分成正常组、OA组、正常＋FCM组及OA＋FCM组，其中正常组和OA组均加入完全高糖培养基，正常＋FCM组和OA＋FCM组均加入FCM，对各组细胞进行形态学观察，于第14天将各组软骨细胞予Ⅱ型胶原（COL2）免疫组化染色及油红O染色，同时应用Western blot技术检测各组软骨细胞中COL2、聚蛋白多糖（Acan）和基质金属蛋白酶(MMP-13)的表达情况。结果：①COL2免疫组化染色：OA组软骨细胞内的COL2免疫组化染色平均吸光度比正常组显著降低(P<0.05)。与OA组相比，正常+FCM组软骨细胞内的COL2免疫组化染色平均吸光度无明显差异，而OA+FCM组显著降低(P<0.05)。与正常+FCM组相比，OA+FCM组软骨细胞内的COL2免疫组化染色平均吸光度显著降低(P<0.05)。②油红O染色：OA组软骨细胞内的油红O染色平均吸光度与正常组相比差异无统计学意义。与OA组相比，正常+FCM组和OA+FCM组软骨细胞内的油红O染色平均吸光度均有增高(P<0.05)，但OA+FCM组增高更为显著(P<0.05)。③Western blot结果：OA组COL2、Acan的表达水平低于正常组(P<0.05)，而MMP-13的表达高于正常组(P<0.05)。与OA组相比，正常＋FCM组COL2、Acan和MMP-13的表达水平无明显差异；相较于正常组及正常+FCM组，OA＋FCM组COL2、Acan的表达水平降低(P<0.05)，而MMP-13的表达水平增高(P<0.05)。结论：OA膝FCM能使正常及OA软骨细胞形态上发生脂肪样变，并通过代谢途径提高软骨细胞MMP-13的表达，加速软骨细胞COL2、Acan的降解，且对OA软骨细胞的破坏更为显著。 |
| 英文摘要: |
| Objective: To observe the effect of human knee infrapatellar fat pad with osteoarthritis (OA) on normal and chondrocytes. Methods: Twenty-four cases of human knee infrapatellar fat pads and cartilages were provided by orthopedics operating room, including 12 cases of acute knee injury, and 12 cases of OA undergoing knee joint replacement. The fat pads were cut and stained with hematoxylin-eosin (HE) staining, and subjected to immunohistochemical staining with leptin. The OA fat pads were used to prepare fat conditioned medium (FCM). The normal chondrocytes were isolated from the integrate specimens of acute knee injury. The OA chondrocytes were isolated from the knee joint cartilage in OA. The cells were cultured and identified in vitro, and were randomly divided into four groups: normal chondrocytes group, OA chondrocytes group, normal chondrocytes + FCM group, OA chondrocytes + FCM group. The normal chondrocytes group and OA chondrocytes group were added with full Dulbecco's Modified Eagle's Medium (DMEM) medium, and the normal chondrocytes + FCM group and OA chondrocytes + FCM group were added with FCM. Each group was observed under a microscope. Cells were stained with type Ⅱ collagen (COL2) immunohistochemical staining and oil red O staining after 14 days. Western blotting was used to examine the expression of COL2, aggrecan, and MMP-13. Results: (1) COL2 immunohistochemical staining: The average absorbance of COL2 immunohistochemical staining in OA group was significantly lower than that in the normal group (P<0.05). As compared with OA group, the average absorbance of COL2 immunohistochemical staining showed no significant difference between OA group and normal + FCM group (P>0.05), and that in OA + FCM group was significantly lower than in OA group (P<0.05). As compared with normal + FCM group, the average absorbance of COL2 immunohistochemical staining in OA + FCM group was significantly decreased (P<0.05). (2) Oil red O staining: The average absorbance of oil red O staining showed no significant difference between OA group and normal group (P>0.05). As compared with OA group, the average absorbance of oil red O staining in normal + FCM group and OA + FCM group was significantly increased, with more significant increase in OA + FCM group (P<0.05). (3) Western blotting showed that the expression of COL2 and aggrecan in the OA chondrocytes group was significantly lower than that in the normal chondrocytes group (P<0.05), but the expression of MMP-13 was significantly higher in the OA chondrocytes group than in the normal chondrocytes group (P<0.05). There was no significant difference in levels of type Ⅱ collagen, aggrecan and MMP-13 between OA chondrocytes group and normal chondrocytes + FCM group (P<0.05). As compared with normal group and normal + FCM group, the expression of type Ⅱ collagen and aggrecan in OA chondrocytes + FCM group was significantly reduced (P<0.05), but the expression of MMP-13 was significantly increased (P<0.05). Conclusions: FCM of OA can induce fatty degeneration of the normal and OA chondrocytes, and it also increases the expression of MMP-13 and decreases the expression of COL2 and aggrecan in chondrocytes through metabolic pathway. Moreover, the damage in OA chondrocytes is more significant. |
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