甲状旁腺激素对髁突软骨细胞增殖、分化的影响

Effects of parathyroid hormone on condylar chondrocyte proliferation and differentiation

目的研究甲状旁腺激素(PTH)在持续性或间歇性作用下对大鼠髁突软骨细胞增殖、分化的影响。方法将分离后的雌性大鼠髁突软骨细胞根据PTH作用方式分为PTH持续应用组、PTH间歇应用组和空白对照组,MTT法检测不同PTH刺激方式对髁突软骨细胞增殖的影响,茜素红染色检测培养后第6、14天各组细胞矿化结节形成情况。提取各组成骨诱导培养第6、14天后细胞总RNA或总蛋白,碱性磷酸酶(ALP)检测试剂盒检测ALP活性,Western blot和Real-time PCR法检测软骨细胞增殖、分化及骨质形成相关蛋白(BSP、BMP2、OCN)和基因(COL2a1、COL10a1、RUNX2、OSX)mRNA的表达水平。结果第二代培养细胞爬片Ⅱ型胶原免疫组化染色结果均为阳性,表明培养细胞为髁突软骨细胞。髁突软骨细胞的增殖能力PTH持续应用组明显高于PTH间歇应用组和空白对照组(P<0.05);PTH持续应用组形成矿化结节数量最少。结论持续性应用PTH能促进髁突软骨细胞增殖,抑制其成熟分化;间歇性应用PTH促进髁突软骨细胞分化及骨质形成,抑制其增殖。

Objective To investigate the effect of parathyroid hormone(PTH)on proliferation and hypertrophic differentiation of rat condylar chondrocytes under persistent or intermittent effects.Methods The isolated female rat condylar chondrocytes were divided into PTH continuous application group,PTH intermittent application group and blank control group according to the mode of PTH action.MTT assay was used to detect the effect of different PTH stimulation methods on the proliferation of condylar chondrocytes.The formation of mineralized nodules in each group was detected by alizarin red staining on the 6th and 14th day after culture.After the 14th day,total RNA or total protein,alkaline phosphatase(ALP)detection kit was used to detect ALP activity,and Western blot and Real-time PCR were used to detect chondrocyte proliferation,differentiation and bone formation related proteins(BSP,BMP2,OCN)and gene(COL2a1,COL10a1,RUNX2,OSX)mRNA expression levels.Results The second-generation cultured cell slides were positive for immunohistochemical staining of typeⅡcollagen,indicating that the cultured cells were condylar chondrocytes.The proliferative capacity of condylar chondrocytes in the PTH continuous application group was significantly higher than that in the PTH intermittent application group and the blank control group(P<0.05).The PTH continuous application group formed the least number of mineralized nodules.Conclusions Continuous PTH administration enhance condylar chondrocyte proliferation and suppress simultaneously condylar chondrocyte differentiation,whereas intermittent PTH administration promote condylar chondrocyte differentiation and bone formation and inhibit its proliferation.