蛇床子素对成骨样细胞UMR106增殖和分化的影响

Effects of osthole on proliferation and differentiation of osteoblast-like cell line UMR 106

目的:以在某种程度上可反映成骨细胞增殖的成骨样细胞UMR106为靶细胞,考察蛇床子素对其细胞形态、增殖率和分化程度的影响。方法:实验于2003-03/2004-03,在中国人民武装警察部队医学院药物化学教研室药物活性筛选室进行。以大鼠成骨肉瘤细胞系UMR106为靶细胞,以雌二醇1×10-8mol/L为阳性对照,同时设空白对照组,蛇床子素干预组分为1×10-8mol/L,1×10-7mol/L,1×10-6mol/L3个浓度组。①调整细胞浓度为4×107L-1,接种于12孔培养板上。48h后,倒置显微镜下观察细胞形态变化。②调整细胞浓度为2×107L-1,接种于96孔培养板,采用四唑盐法测定各孔的吸光度,计算平均增殖率。③调整细胞浓度为2×107L-1,接种于24孔培养板,磷酸对硝基苯基质动力学法测定细胞内外碱性磷酸酶活性。上述实验每组均设8个复孔。结果:①培养基中加入蛇床子素培养48h后,与对照组相比UMR106细胞数量明显增多,核分裂期多见,1×10-6mol/L,1×10-7mol/L组可见细胞重叠生长,分泌基质堆积明显。②UMR106细胞经蛇床子素处理48h后,相对于对照组,1×10-6mol/L组增殖率为52%,1×10-7mol/L组为37%,1×10-8mol/L组为16%。3个给药组与对照组相比差异均有显著性(t=37.36,14.94,6.81,P<0.01)。③UMR106细胞经蛇床子素1×10-6mol/L和经雌二醇1×10-8mol/L处理24h和48h后,细胞内碱性磷酸酶活性均显著高于对照组犤(825.17±12.34),(775.16±8.67),(715.14±14.17)μkat/g;(2415.48±15.84),(2355.47±5.67),(2285.49±7.67)μkat/g;t=16.56,10.22;20.89,34.90,P<0.01犦。④UMR106细胞在浓度为1×10-6,1×10-7mol/L的蛇床子素及雌二醇10-8mol/L培养24h后,细胞外碱性磷酸酶活性显著高于对照组犤(781.66±11.50),(733.15±15.34),(728.81±9.84),(652.80±11.34)μkat/g;t=22.56,11.91,14.32,P<0.01犦。结论:蛇床子素可剂量依赖地促进UMR106细胞的增殖,刺激UMR106细胞的碱性磷酸酶活性,提示其可能具有直接促进成骨细胞增殖、分化的作用。

AIM： To regard the osteoblast-like cell line UMR 106 that can reflect the proliferation of osteoblast to some extent as target cell, and study the effects of osthole on the morphology, proliferation and differentiation of osteoblast-like cell line UMR 106. METHODS： The experiment was conducted at the screening room of activity of drug, Staff Room of Pharmacology and Chemistry, Medical Collage of Chinese People＇s Armed Police Forces from March 2003 to March 2004. Osteogenic sarcoma cell line UMR106 of rats was considered as target cell, and taking aquadiol 1×10^-8 mol/L as positive control. Meanwhile, the blank control group and osthole interventional group were assigned into 1×10^-8 mol/L, 1×10^-7 mol/L, 1×10^-6 mol/L groups. ①Cell concentration was adjusted into 4×10^-7L^-1, the cells were inoculated on 12- well plates. After 48 hours, morphologic change of cells was observed under invert microscope. ②The cellconcentration was adjusted into 2×10^7L^-1, and the cells were inoculated on the 96-well plates. Absorbance （A） of every hole was detected with MTY method and the rates of average proliferation were calculated. ③The UMR106 cells were plated on 24-well plates at a density of 2×10^7L^-1 and the activities of alkaline phosphatase （ALP） in and out of cells were detected by PNPP approach. Every abovementioned group included 8 wells. RESULTS： ①The cells were treated with osthole for 48 hours, as compared with control group the numbers of the UMRI06 cell and the karyokinetic phase increased. The overlapping cells and the stack significantly matrix were observed when the cells were treated with osthole of 1×10^-6 mol/L or 1×10^-6mol/L. ②The cells were treated respectively with osthole of 1×10^-6mol/L, 1×10^-7mol/L and 1×10^-5 mol/L for 48 hours, the rate of proliferation was respectively 52%, 37% and 16% and showed a significant difference as compared with the control group （t=37.36, 14.94, 6.81, P 〈 0.01 ）. ③The cells were treated respectively with osthole of 1×10^-6 mol/L and estradiol of 1×10^-8 mol/L for 24 hours and 48 hours, as compared with the control group, the activities of ALP in cells increased significantly [（825.17 ±12.34）, （775.16±8.67）, （715.14±14.17） μkat/g and （2 415.48±15.84）, （2 355.47±5.67）, （2 285.49±7.67） μkat/g, t=16.56,10.22 and 20.89, 34.90, P 〈 0.01]. ④The cells were treated respoctively with osthole of 1×10^-6 mol/L, 1×10^-7mol/L and estradiol of 1×10^-8 mol/L for 24 hours, the activities of ALP out of cells increased significantly as compared with the control group [（781.66±11.50）, （733.15 ± 15.34）, （728.81±9.84）, （652.80±11.34）μ kat/g; t=22.56, 11.91, 14.32, P 〈 0.01]. CONCLUSION： Osthole can accelerate dose-dependently the proliferation of UMR106 cells and stimulate the activity of ALP of esteoblast-like cells UMR 106. It is suggested that the osthole may improve directly the proliferation and differentiation of osteoblasts.