C反应蛋白激活原纤毛抑制颅骨成骨细胞的增殖和分化  

C-reactive protein inhibits the proliferation and differentiation of calvarial osteoblasts primary via cilia

作　　者：许洁[1] 刘杰[1] 汪长东[1] XU Jie;LIU Jie;WANG Changdong(Department of Biochemistry and Molecular Biology,Chongqing Medical University,Center for Oncology and Molecular Medicine,Basic Medical College,Chongqing 400016,China)

机构地区：[1]重庆医科大学生物化学与分子生物学教研室,肿瘤与分子医学中心,基础医学院,重庆400016

出　　处：《中国骨质疏松杂志》2021年第8期1163-1168,共6页Chinese Journal of Osteoporosis

基　　金：重庆市研究生科研创新项目(CYS19204);2020年重庆市渝中区科研计划第一批项目(20200112)。

摘　　要：目的研究C反应蛋白(C-reactive protein,CRP)对原代培养SD大鼠乳鼠颅骨成骨细胞(rat calvarial osteoblasts,ROB)增殖及成骨分化的影响。方法取SD乳鼠颅骨组织用胰蛋白酶和胶原蛋白酶Ⅰ消化得到原代ROB,随后将ROB用α-MEM培养基培养,传代3次后用于后续实验。在对照组和5 mg/mL CRP的处理组中,成骨分化培养基诱导3 d进行免疫荧光检测两组Ki-67和acetylated-α-tubulin和γ-tubulin表达情况;同时,蛋白质印迹分析osteopontin(OPN)、alkaline phosphatase(ALP)、acetylated-α-tubulin和γ-tubulin的表达;成骨分化培养基诱导21 d茜素红染色检测成骨基质矿化结节量。结果免疫荧光显示,5 mg/mL CRP抑制成骨细胞增殖,与对照组差异有统计学意义(P<0.001);蛋白质免疫印迹和茜素红染色显示,处理组OPN和ALP表达降低(P<0.01),矿化结节生成数量减少(P<0.001),与对照组比较差异均有统计学意义;免疫荧光结果显示,细胞原纤毛长度增加(P<0.001),免疫印迹结果显示acetylated-α-tubulin和γ-tubulin蛋白的表达均升高(P<0.001)。结论5 mg/mL CRP显著抑制ROB增殖与成骨分化矿化并且显著激活细胞原纤毛。CRP为炎症因子典型代表,这些数据表明炎症因子激活细胞原纤毛发挥其抑制成骨细胞的增殖与分化作用,提示抑制原纤毛或阻断炎症因子的释放可以治疗炎症引起的骨丢失疾病。Objective To investigate the effect of C-reactive protein(CRP)on the proliferation and osteoblast differentiation of rat calvarial osteoblasts(ROB)in Sprague Dawley(SD)rats.Methods ROB was obtained from the skull tissue of SD suckling rats by digestion with trypsin and collagenase I.ROB was then cultured inα-MEM medium and subcultured 3 times for the follow-up experiment.The expression of Ki-67 and acetylated-α-tubulin,γ-tubulin were detected by using immunofluorescence in ROB with or without 5 mg/mL of CRP after 3 days induction.The expression of osteopontin(OPN),alkaline phosphate(ALP),acetylated-α-tubulin,andγ-tubulin proteins were analyzed with Western blotting.Alizarin red staining was used to detect the amount of matrix mineralized nodules after 21 days induction.Re sults Immunofluorescence result showed that 5 mg/mL of CRP inhibited the proliferation of osteoblasts,which was significant different from that of control group(P<0.001).Western blotting and Alizarin red staining result showed that the expression of OPN and ALP decreased in the treated group(P<0.01),and the number of mineralized nodules decreased(P<0.001),which were statistically significant compared with those in the control group.Immunofluorescence result showed that both the length of primary cilia and the expression of acetylated-α-tubulin andγ-tubulin increased(P<0.001).Conclusion CRP at a concentration of 5 mg/mL significantly inhibits the proliferation and osteoblast differentiation of ROB and activates the expression of cilia.CRP is a typical representative of inflammatory factors.These data indicate that inflammatory factors activate the primary cilia to inhibit the proliferation and differentiation of osteoblasts,suggesting that inhibiting primary cilia or blocking the release of inflammatory factors might be a possible treatment for inflammatory bone loss.

关 键 词：C反应蛋白 颅骨细胞 增殖 分化 炎症 

分 类 号：Q257[生物学—细胞生物学]