破骨细胞酸性磷酸酶染色后快速半定量分析  

Fast semi-quantitative analysis of TRAP staining for osteoclast

作　　者：何友华[1] 童国军[1] 李明翰 李威[1] 许伏龙 杨德鸿[1] HE You - hua;TONG Guo -jun;LIMing - han;LI Wei;XU Fu - long;YANG De - hong(Department of Spine Orthopaedics, the Southern Medical University South Hospital, Guangzhou 510515, Guangdong , China)

机构地区：[1]南方医科大学南方医院脊柱骨科,广东广州510515

出　　处：《广东医学》2018年第12期1765-1768,共4页Guangdong Medical Journal

基　　金：国家自然科学基金资助项目(编号:81272043)

摘　　要：目的寻求合适的溶剂,溶解破骨细胞的抗酒石酸酸性磷酸酶(TRAP)染色产物,筛选溶液的最适检测波长,检测溶液吸光度值。比较该方法与TRAP活性检测试剂盒法相关性,并评价该快速半定量分析法的稳定性与可靠性。方法提取C57BL/6小鼠骨髓来源巨噬细胞(BMMs),以1×10~4·mL^(-1)均匀种入胶原I包被处理24孔板,随机分为4组:10 nmoL/L PTH(1-34),100 nmoL/L PTH(1-34),1μmoL/L PTH(1-34),10μmoL/L PTH(1-34),给予甲状旁腺素[PTH(1-34)]刺激4 h后换成含10%胎牛血清完全培养基培养44 h,以4 h/44 h为1个周期,培养8 d后,进行常规破骨细胞TRAP染色,筛选合适的溶剂溶解,最终使用冰醋酸溶解TRAP原位染色产物,超声粉碎取上清,选取合适的波长检测吸光度(OD)值,此为冰醋酸溶解定量法。另一检测手段为目前常规使用的酶活性检测试剂盒法,试比较冰醋酸溶解定量法与酶活性检测试剂盒法两者的相关性。结果常规破骨细胞原位TRAP染色后,筛选出冰醋酸的溶解效果最优。溶解后测定波长在550 nm左右吸收峰理想,并排除溶剂冰醋酸本身的干扰。随着PTH(1-34)浓度增高,表现出破骨效应的增加,TRAP的活性增加,两者的OD值相应增加。冰醋酸溶解定量法与酶活性检测试剂盒法存在显著正相关,Pearson相关系数r=0.986(P<0.05)。结论采用冰醋酸溶解定量法,并用550 nm波长测定OD值,可实现破骨细胞TRAP染色后的快速半定量分析。Objective To select a suitable solvent for dissolving osteoclast tartrate resistant acid phosphatase stai- ning （the TRAP in situ staining） ; to explore the optimal wavelengths of light absorption; and to compare with tartrate re- sistant acid phosphatase detection kit method, thus evaluate the stability of fast semi - quantitative analysis. Methods Seeding the bone marrow - derived macro - phages （BMMs） in 24 wells pretreated with plate collagen I. The cells were extracted from C57BL/6 mice, then divided into 4 groups, 10nmoL/L PTH （ 1 - 34） , 100 nmoL/L PTH （ 1 - 34） , 1 μmol PTH （ 1 - 34）/L and 10 μmol/L PTH （ 1 - 34） groups. The cells were incubated with PTH for 4 hours then replaced with a medium containing 10% fetal bovine serum for 44 hours, 4 h/44 h as a cycle. Eight days later, regular TRAP in situ staining was performed. Suitable solvents were screened for TRAP. Acetic acid was applied for final solved, with ultrason- ic grinding. Supernatant was collected for assessment of OD value with a suitable absorbance wavelength, and compared with tartrate resistant acid phosphatase kits. Results After TRAP in situ staining, acetic acid was chose as the optimized solvent. The 550 nm absorbance wavelength was ideal, when eliminating the interference of acetic acid. As the increasing of the concentration of PTH（ 1 -34） , the activity of osteoclasts increased, and the OD value increased as the same. The 01） value of TRAP in situ staining solution and the OD value of tartrate resistant acid phosphatase detection kit method had significant correlation （ r = 0. 986, P 〈 0. 014）. Conclusion Dissolving the TRAP in situ staining with acetic acid, using 550nm absorbance wavelength can realize osteoclast rapid semi -quantitative analysis after dyeing.

关 键 词：TRAP原位染色 冰醋酸 半定量分析 破骨细胞 甲状旁腺素 

分 类 号：R580[医药卫生—内分泌]