摘要
目的研究蛇床子素(osthole,OST)对破骨细胞形成和骨吸收的影响,并初步分析其分子机制。方法体外构建破骨细胞诱导培养体系,通过抗酒石酸酸性磷酸酶染色(tartrate resistant acid phosphatase,TRAP)法、骨吸收陷窝扫描电镜观察鉴定成熟破骨细胞,采用CCK-8 (cell counting kit-8)法筛选无细胞毒性的药物浓度组,使用细胞骨架荧光染色法观察OST对其形态的影响。使用骨吸收陷窝甲苯胺蓝染色进行面积定量分析,并统计吖啶橙荧光染色后凋亡破骨细胞的比例,采用荧光定量PCR法测定OST对NFATc1等相关破骨基因表达的影响。结果体外成功构建破骨细胞培养体系,通过CCK-8法筛选出10-6、10-5mol/L两组药物浓度。OST可使破骨细胞肌动蛋白环变细,伪足和褶皱区消失。OST 0、10-6、10-5mol/L组TRAP染色阳性数目分别为104. 6±4. 5、80. 4±3. 7、58. 2±3. 1,融合指数分别为(44. 3±3. 3)%、(20. 3±0. 9)%、(12. 6±0. 6)%,各浓度组组间比较,差异均有统计学意义(P<0. 05);该3组诱导的破骨细胞产生的骨陷窝数目为41. 67±1. 16、34. 01±2. 65、26. 33±4. 16,骨陷窝面积(μm2/片)分别为1 445 942. 0±164 150. 0、904 080. 8±47 346. 0、641 049. 1±1 993. 0,各浓度组组间比较,差异均有统计学意义(P<0. 05);该三组破骨细胞凋亡率分别为:26. 3%、30. 1%、37. 2%,OST组凋亡率较对照组明显升高,且差异有统计学意义(P<0. 05),但两浓度组之间差异无统计学意义(P> 0. 05)。RANKL诱导后NFATc1、CTSK、MMP-9、TRAP、INTE-BETA3、C-SRC的mRNA表达量分别为空白组的2. 147±0. 246、30. 5±1. 643、43. 54±2. 908、9. 116±0. 392、6. 664±0. 395、4. 131±0. 408倍,与空白组相比表达量均明显升高,差异具有统计学意义(P<0. 01);OST干预后,各组表达量均受到明显抑制,OST组表达量与RANKL对照组相比差异也均具有统计学意义(P<0. 05)。除了NFATc1外,两浓度组组间比较,差异均有统计学意义(P <0. 05)。结论 OST可以通过调控NFATc1等破骨基因表达抑制破骨细胞的分化。
Objective To study the effect of osthole （OST） on osteoclast formation and bone resorption runetion, and to verify its molecular mechanism. Methods To construct the osteoclast-induced culture system in vitro. To observe the osteoclast ceils by chimeric acid electrophoresis tartrate resistant acid phosphatase （TRAP） method and observe the osteoclasts by scanning electron microscopy. The effect of OST on the cytoskeleton morphology was observed by fluorescence staining. Thearea was quantitatively analyzed by toluidine blue staining of bone resorption lacunae. The ratio of apoptotic osteoclasts was observed by acridine orange staining, qPCR was used to determine the effect of OST on the expression of NFATcl and other related osteoclasts. Results The osteoclast culture system was successfully constructed in vitro. The concentration of 10-6 mol/L and 10-5 mol/L groups was selected by CCK-8 method. OST could make the actin ring of osteoclast become thin, the pseudo foot and the fold area disappeared. The number of TRAP staining positive cells in group OST 0, 10 6, and 10-5 mol/L were 104. 6±4. 5, 80. 4±3.7, and 58.2±3.1. The indexes were （44. 3±3.3）%, （20. 3±0. 9）%, and （ 12. 6±0. 6% ） respectively. The differences were statistically significant （P〈0. 05 ） in each group. The number of osteoclasts induced-lacuna by the three groups was 41.67±1.16, 34. 01±2. 65, 26. 33±4. 16, and the area of bone lacunae （μm2/slices） were 1 445 942. 0±164 150. 0, 904 080. 8±473 46. 0, 641 049. 1±1 993.0 respectively. The difference were statistically significant （P〈0. 05） in each group. The rate of apoptosis in the three groups of osteoclasts were 26. 3%, 30. 1%, 37.2%, and the rate of apoptosis in group OST was significantly higher than that of the control group （P〈0. 05）, but there was no significant difference between the two concentration groups （P〉0. 05）. The expression of mRNA in NFATcl, CTSK, MMP-9, TRAP, INTE-BETA3, and C-SRC were 2. 147±0. 246, 30. 5± 1. 643, 43.54±2. 908, 9. 116±0. 392, 6. 664± 0. 395, 4. 131 ± 0. 408, respectively, and the difference was significantly higher than that in the blank group （P〈0. 01 ）. After using OST, the expression of OST group was significantly inhibited compared with RANKL Control group （P〈0. 05）. Except for NFATcl, the difference between the two concentration groups was statistically significant （P〈0. 05 ）. Conclusion OST can inhibit osteoclast differentiation, and it has the potential to develop anti-osteoporosis drugs.
作者
王礼宁
马勇
郑苏阳
郭杨
潘娅岚
王磊
顾鸣
孙杰
WANG Li-ning;MA Yong;ZHENG Su-yang;GUO Yang;PAN Ya-lan;WANG Lei;GU Ming;SUN Jie(Institute of Traumatology,Nanfing University of Chinese Medicine,New Technology on Trauma Repairment and Reconstruction Laboratory,Nanjing University of Chinese Medicine,Nanjing 210023,China;Department of Orthopaedics,Hospital Affiliated to Nanjing University of Chinese Medicine,Nanjing 210029,China)
出处
《中华骨质疏松和骨矿盐疾病杂志》
CSCD
北大核心
2018年第5期475-483,共9页
Chinese Journal Of Osteoporosis And Bone Mineral Research
基金
国家自然科学基金面上项目(81473692)
南京中医药大学骨伤修复与重建新技术实验室专项资金项目
