骨保护素对破骨细胞的影响程度分析
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骨保护素对破骨细胞的影响程度分析
[摘要] 目的 通过分析比较不同浓度的骨保护素(osteoprotegerin,OPG)对破骨细胞(osteoclasts,OC)活性的影响程度来研究2者在生理活性及生理功能之间的相关性。方法
本次实验应用6周龄的雌性小鼠模型,添加不同浓度的骨保护素后通过抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRAP)染色、细胞骨架F-actin染色及骨吸收陷窝的检测,观察OPG与OC之间的相关性。结果 OC培养1h内即贴壁生长,并为展开且无大的多核OC存在。培养至第5天,较小的单核细胞逐渐开始相互融合,形成多核细胞且特征明显。OPG处理3 d后,对照组细胞形态整体性良好,利用观察比较。随着OPG 浓度的增大,实验组的多核细胞数逐渐减少,多核细胞数随着骨保护素(OPG)浓度的逐渐增加而减少,2者间呈现负相关的关系(r=0.516,P<0.05)。且当骨保护素(OPG)浓度为50μg/L 时,视野内仅存在少量多核细胞,模糊可见。培养结束后,OPG组多核细胞数随着OPG浓度的增大而逐渐减少,且OPG浓度为50ng/mL时仅有少量多核细胞可见;OPG的浓度为20、50 μg/L时,OPG组的OC数较对照组显著减少(P<0.05),而当OPG的浓度为100μg/L时,OPG组的OC数较对照组呈极显著减少(P<0.01);OPG实验组的骨吸收陷窝的面积、密度和减弱程度均与OPG的浓度呈正相关(r=0.459,P<0.05),且面积比较具有显著性差异(P<0.05)。结论 不同浓度的OPG对破骨细胞的影响程度不同,且OPG的浓度越大其抑制性越强。
[关键词] 骨保护素;破骨细胞;吸收陷窝
[中图分类号] R3 [文献标识码] A
Analysis of the influence degree of osteoprotegerin on osteoclasts
LIU Jian1,SONG Hui2
(1.Department of orthopedics, Shandong Hospital of Shengli oil field in Dongying City,Dongying
257000,China.2. Department of oral and maxillofacial surgery, Shandong University Jinan
250000,China.)
[Abstract]: Objective Effects of different concentrations of OPG (osteoprotegerin, OPG) on
osteoclasts (osteoclasts, OC) the degree of influence activity, provide a theoretical basis for further
research OPG and physiological functions of OC. Methods 6-week-old female mice were cultured in vitro on the basis of adding different levels of OPG, by TRAP staining, F-actin cytoskeleton
staining and resorption correlation detection nest was observed between OPG and OC. Results
OC culture that is adherent growth within 1h, and for the launch of a large multi-core and no CO
present. Cultured for 5 days, the smaller monocytes gradually began to fusion with each other to
form multinucleated cells and distinctive characteristics. OPG treatment after 3 d, cell morphology
control group overall good use of observation and comparison. With OPG concentration increases,
the number of cells in the experimental group multicore gradually reduce the number of cells with
increasing multicore osteoprotegerin (OPG) concentration decreased, showing a negative
correlation between the two relationships(r=0.516,P<0.05). And when osteoprotegerin (OPG)
concentration of 50ng · mL-1, there is only a small amount of multinucleated cells within the field
of vision, blurred visible. After observing the culture, OPG Group polynuclear cells with OPG
concentration increases gradually reduced, and OPG concentration of 50ng · mL-1 is only a few
multinucleated cells visible; concentration of OPG was 20,50 μg · L-1 when, OC number OPG
group significantly decreased significantly reduced [OC than the control group, is the result of
comparing what time and under what time come? ] (P <0. 05), and when the concentration of OPG
was 100 μg · L-1 时, OC number OPG group than in the control group showed significant
reduction [and who compare? ] (P <0 01.); By X2 test and Spearman rank correlation analysis, the
experimental group OPG bone resorption lacunae area, density and decrease the degree of
concentration of OPG were positively correlated, and the area was a significant difference (X2 =
15.119, r = 0.459, P <0. 05). Conclusion Effects of different concentrations of OPG OPG
observed for OC formation and activation, can provide a theoretical basis for further study of OPG
and physiological functions of OC.
[Key words]:osteoprotegerin(OPG); osteoclasts(OC);Resorption pits
破骨细胞(osteoclasts,OC)来源于特定的破骨前体(osteoclast precursors,OCPs),在骨吸收功能中维持至骨代谢平衡,且与全身或局部性的骨质疏松密切相关[1]。研究表明[2],骨营养不良的最主要原因为骨吸收程度大于骨在建程度,这也间接说明了破骨细胞对骨质的影响相对较大。此外,OC活性的异常变化可对机体骨骼产生不同程度的病理性变化,如骨质疏松等。近些年发现[3]OPG可以通过OPG/RANK/RANKL机制抑制OC的分化、活化成熟并诱导OC的凋亡,且有望成为治疗多种骨代谢疾病的理想药物。但当前国内外对于骨保护素与破骨细胞之间的活化影响成对的报道较少,且其中关于活化和生成的机理尚不明确。故本试验通过对不同浓度的骨保护素对破骨细胞的生成和活化程度的影响来研究2者在生理活性及生理功能之间的相关性。
1 材料与方法
1.1 主要实验仪器与试剂
超净工作台(北京精密仪器厂);PS-9000705超纯水装置(美国 LABCONCO 公司);CO2温培养箱(Heraeus公司);倒置相差显微镜(Olympus公司:IX 7000) ;扫描电子显微镜(日电,JEOL JSM-T300)。抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRAP)染色试剂盒购自美国Sigma公司;α-MEM(Gibco);胎牛血清购自杭州四季青生物工程有限公司);1600锯式切片机(Leica)、12、48孔培养板(兴万Corning);牛皮质骨片片(直径50 μm,IDS);骨保护素(PeproTech A)。
1.2 OC分离与培养
OC分离在参考付应宵[4]的分离方法上做出一定的改进。本实验选取6周龄的雌性ICR小鼠,使其脱颈窒息而死,将小鼠的前肢肱骨及后肢长骨在无菌环境下进行分割分离,取骨髓细胞并于200 g/min下离心5 min,取沉淀物。重悬于含α-MEM的培养液中(含2mmol/L
L-谷氨酰胺,10%FBS、100 IU/mL 青霉素、100 mg/L链霉素),按操作规程要求将其分别接种于12孔培养板和48孔培养板,其中在48孔板中部分放置牛骨片,同时在5%CO2饱和湿度下的环境下培养24 h。贴壁细胞用37℃含0.05%吐温-20的pH7.4的磷酸盐缓冲液反复冲洗,继续培养,每天更新培养液。如此反复操作3天。第4天,其中对照组加入0 μg/L的OPG,实验组加入10、20、50和100 μg/L的OPG,按上述条件继续培养3天后,进行检测。
1.3 染色处理
经骨保护素(OPG)处理3 天后,将细胞培养板取出,用含0.05%吐温-20的pH7.4的磷酸盐缓冲液冲洗3次,然后以4%多聚甲醛固定10 min,使用TRAP试剂盒进行检测。将玻片取出,用枸橼酸/Acetone溶液进行固定后进行染色,使其在37℃的环境下孵育60 min,清洗、晾干、封片,倒置显微镜观察。