摘要
目的:观察能诱导体外培养的破骨细胞凋亡的骨康含药血清对新生大鼠成骨细胞凋亡的抑制作用。方法:实验于2003-01/2003-06在广州中医药大学第一附属医院骨科实验室完成。①选用1d龄雌性SD大鼠3只,用于分离原代成骨细胞;6月龄SD雌性大鼠24只用于制备含药血清。②6月龄SD雌性大鼠随机分成3组,每组8只,分别为骨康方组、罗盖全组和空白对照组(蒸馏水灌胃)。骨康方由淫羊藿、补骨脂、黄芪、熟地、白芍等药物组成,为中药煎剂,含生药量1.43kg/L,按4.8g/kg生药灌胃,相当于临床剂量的10倍。罗盖全(活性维生素D3)按0.084μg/kg灌胃,相当于临床剂量的10倍。各组大鼠分别用上述物质灌胃,每次17.5mL/kg,2次/d,间隔5h,连续灌胃3d,最后1次灌胃后2h腹主动脉取血,离心获取含药血清。将传代的成骨细胞分别加入含药血清培养7d时,加入肿瘤坏死因子α,剂量为30μg/L,分别于12和24h后取出长有成骨细胞的盖玻片,经吖啶橙染色,置Nikon荧光显微镜下直接观察拍照。③成骨细胞的鉴定,包括形态学观察和碱性磷酸酶染色和矿化结节茜素红染色观察。结果:①在用空白血清培养7d的成骨细胞中加入肿瘤坏死因子α,12h时可见到成骨细胞出现凋亡征象,24h时出现典型的细胞凋亡形态。②在用骨康血清和罗盖全血清培养7d的成骨细胞可部分对抗肿瘤坏死因子α所致的成骨细胞凋亡作用,中药骨康血清和罗盖全血清对成骨细胞凋亡的抑制作用相似。结论:①肿瘤坏死因子α可诱导成骨细胞凋亡。②用中药骨康含药血清培养7d的成骨细胞可部分对抗肿瘤坏死因子α对其的损伤,提示中药骨康可通过阻滞肿瘤坏死因子α诱导的成骨细胞凋亡,发挥防治骨质疏松的作用。
AIM: to observe the effect of Gukang drug serum, which can induce in vitro cultured osteoclast apoptosis, in inhibiting the osteoblasts apoptosis of newborn rats. METHODS: The experiment was completed in the orthopedic laboratory of the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine from January to June 2003. ① Three female SD rats of 1-day-old were chosen to separate primary ostoblasts; Twenty-four female SD rats of 6-month-old were applied to produce drug serum. ② The 6-month-old female SD rats were randomly divided into 3 groups with 8 rats in each group: Gukang fang group, Rocaltrol group and blank control group (grastric perfusion of distilled water). Gukang fang was a Chinese herb decoction consisted of Korean epimedium herb, malytea scurfpea fruit, membranous milkvetch root, prepared rehmannia root, white peony root, etc., containing crude drug of 1.43 kg/L, gastric perfusion of crude drug was given according to 4.8 g/kg, which was equal to 10 times of the clinical dosage. Rocaltrol (activated vitamin D3) was gastrically perfusion according to 0.084 μg/kg, which was equal to 10 times of the clinical dosage. Rats in each group were treated with gastric perfusion of above drugs, 17.5 mL/kg for each time, twice a day with an interval of 5 hours for 3 continuous days. Blood samples were drawn from abdominal aorta 2 hours after the last gastric perfusion, drug serum was obtained with centrifugation. The passages of osteoblasts were cultured with drug serum for 7 days, and then tumor necrosis tumor alpha (30 μg/L) was added, the cover glasses with osteoblasts were taken out afther 12 and 14 hours respectively; After staining of acridine orange, they were observed and photographed under Nikon fluorescence microscope. ③ The identification of osteoblasts included morphological observation, staining of alkaline phosphatase and staining of mineralized tuberosity alizarin bordeaux observation. RESULTS: ①After tumor necrosis tumor alpha was added to the osteoblasts, which was cultured in blank serum for 7 days, apoptosis was observed at 12 hours, and typical apoptosis morphology occurred at 24 hours. ② The osteoblasts cultured in Gukang serum and Rocaltrol serum for 7 days, could partly suppress the tumor necrosis tumor alpha induced osteoblasts apoptosis, the inhibitory effects of Chinese herb Gukang drug serum and Rocaltrol serum on the osteoblasts apoptosis were similar. CONCLUSION: ① Tumor necrosis tumor alpha can induce the apoptosis of osteoblasts. ② The osteoblasts cultured in Chinese herb Gukang drug serum and Rocaltrol serum for 7 days can partly fight against the injury caused by tumor necrosis tumor alpha, it is indicated that the Chinese herb Gukang can plays a role in preventing and treating osteoporosis through suppress tumor necrosis tumor alpha induced osteoblast apoptosis.
出处
《中国临床康复》
CSCD
北大核心
2005年第23期170-172,i004,共4页
Chinese Journal of Clinical Rehabilitation
基金
广东省中医药局资助项目(100044)~~
