续断含药血清对成骨细胞增殖的影响及其细胞毒性检测

Influence of rat serum containing Radlx Dipsaci on the proliferation of osteoblasts as well as the cytotoxic detection

目的:观察不同浓度续断含药血清的细胞毒性及其对成骨细胞增殖的影响。方法:实验于2001-01/07在中山医科大学附属第二医院脐血库、医学研究中心以及中山医科大学放免检测中心完成。①制备大鼠含药血清:取雌性SD大鼠18只,随机分为3组,续断组6只,空白对照组8只,雌激素组4只,按体质量分别给予续断生药、生理盐水、雌激素灌胃3d后取血,离心后取上清液。②根据文献方法分离、培养人的原代成骨细胞。③含药血清细胞毒性的观察:成骨细胞按1.0×108L-1密度接种培养24h,换成无血清培养液24h后,用含不同续断浓度(100%,50%,25%,12.5%,6.25%,3%)的大鼠血清处理细胞。采用直接计数法和四氮唑蓝法观察不同浓度续断含药血清的细胞毒性。④细胞增殖的定量分析:将培养人成骨样细胞用不同组大鼠血清稀释,分为5%,10%和20%续断组,5%,10%和20%雌激素1组,5%,10%和20%空白对照组,5%,10%和20%雌激素2组。采用氚-胸腺嘧啶和四氮唑蓝法检测细胞增殖能力。结果:①续断含药血清细胞毒性的观察结果:直接计数表明含续断血清浓度12.5%和25%时,细胞生长数量最多,四氮唑蓝法测定A值最高。②续断对成骨细胞增殖的影响:氚-胸腺嘧啶掺入法显示10%续断组,10%,20%雌激素1组,各浓度雌激素2组细胞增殖显著高于空白对照组、5%雌激素1组和5%续断组(P<0.01);四氮唑蓝法显示10%,20%续断组,10%,20%雌激素1组,10%,20%雌激素2组细胞增殖显著高于空白对照组、5%雌激素1组和5%续断组(P<0.01)。结论:高剂量续断对成骨细胞增殖产生抑制作用,低剂量无明显影响,中剂量续断对细胞增殖的刺激作用最强。

AIM： To observe the cytotoxicity of rat serum containing Radix Dipsaci at different concentration on proliferation of human osteoblast-like cells. METHODS： The experiment was performed at Umbilical Cord Blood Bank and Medical Research Center of the Second affiliated Hospital of Sun Yat-sen University of Medical Sciences, and the Radioimmunoassay Center of Sun Yat-set University of Medical Sciences from January to July 2001. ①Preparation of the rat serum containing drug： 18 SD male rats were selected and randomly divided into 3 groups with 6 rats in Radix Dipsaci group, blank control group with 8 rats and estrogen group with 4 rats. After being treated with Radix Dipsaci, saline and estrogen, respectively with gastric perfusion according to body mass for 3 days, blood was gained. After contrifugation, superuatant was obtained. ②Human osteoblast-like cells were isolated and cultured according to references. ③Observation on cytotoxicity of rat serum containing Radix Dipsaci： The osteoblasts were planted at 1.0×10^8 L^-1 and cultured for 24 hours. Then they were culture in the medium without any serum for the second 24 hours, At last, they were disposed with the medium contains 100% ,50% ,25%, 12.5% ,6.25% and 3% concentration of Radix Dipsaci rat serum. The cytotoxicity of Radix Dipsaci rat serum was scored by direct cytometry and MTT methods. ④Quantitative analysis on proliferation of human osteoblast-like cells： The cells were disposed with different concentration of rat serum, and divided into 5%, 10% and 20% Radix Dipsaci group, 5%, 10% and 20% estrogen 1 group, 5%, 10% and 20% blank control group with 5%, 10% and 20% estrogen 2 group. The proliferation of cells was tested with ^3H-Tdr and MTT. RESULTS： ①Observational result of cytotoxicity of serum containing Radix Dipsaci： The direct cytometry indicated that the cells had a good proliferation when the medium with 12.5% and 25% concentration of Radix Dipsaci rat serum. MTT results showed that A value was the highest. ②Effect of Radix Dipsaci on proliferation of osteoblast： The result of ^3H-Tdr indicated that the proliferation of human osteoblast-like cells in the 10% Radix Dipsaci group, 10% and 20% estrogen 1 group and estrogen 2 at every concentration of rat serum group was higher significantly than that in the blank control group, 5% estrogen 1 group and 5% Radix Dipsaci group （P 〈 0.01 ）. The MTT showed that the proliferation of cells in the 10% and 20% Radix Dipsaci group, 10% and 20% estrogen 1 group and 10% and 20% estrogen 2 group was higher obviously than that in the blank control group, 5% estrogen 1 group and the 5% Radix Dipsaci group （P 〈 0.01 ）. CONCLUSION： High concentration of Radix Dipsaci has inhibitory effect on the proliferation of osteoblasts, while low concentration of Radix Dipsaci has no effect on the proliferation of cells, and middle concentration of Radix Dipsaci has the strongest effect on the proliferation of cells.