【摘要】 背景:脱氢表雄酮主要来自肾上腺皮质,在相关外周组织中转化为雄激素或雌激素而发挥间接的生物学作用。目的:实验拟验证脱氢表雄酮促进成骨细胞增殖的作用及其机制。设计、时间及地点:分组对照观察,于2006-01/2007-02在上海交通大学附属第六人民医院和上海复旦大学放射医学研究所合作完成。材料:1日龄SD新生大鼠10只,雌雄不拘。脱氢表雄酮为美国Sigma公司产品。方法:体外分离培养SD大鼠成骨细胞,取传1代细胞,分别给予(1×10-5,1×10-7,1×10-9)mmol/L脱氢表雄酮培养72h,以1×10-8mmol/L雌二醇为阳性对照,另设空白对照组。主要观察指标:以细胞形态学和碱性磷酸酶染色进行成骨细胞鉴定。应用光镜、四甲基偶氮唑盐法检测细胞的生长和增殖。茜素红染色观察成骨细胞矿化结节形成功能;同时双抗体夹心ABC-ELISA法测定不同浓度脱氢表雄酮培养液中对骨保护素浓度的影响。结果:①原代培养细胞碱性磷酸酶染色成阳性鉴定为成骨细胞。②不同浓度脱氢表雄酮组成骨细胞增殖能力均有上升,其中以1×10-7mmol/L脱氢表雄酮组最显著(P<0.01),其作用和雌二醇相似(P>0.05)。③不同浓度脱氢表雄酮组碱性磷酸酶的活性均升高,亦以1×10-7mmol/L脱氢表雄酮组最显著(P<0.01),1×10-7mmol/L,1×10-9mmol/L脱氢表雄酮的作用和雌二醇和相似(P>0.05)。④1×10-9mmol/L脱氢表雄酮组单位细胞数的碱性磷酸酶活性高于空白对照组(P<0.05)。⑤不同浓度脱氢表雄酮组矿化面积及矿化面积比均有显著增加(P<0.01),其作用和雌二醇相似。⑥1×10-9mmol/L脱氢表雄酮组培养72h时骨保护素反应最活跃(P<0.01),与成骨细胞增殖能力呈正相关。结论:脱氢表雄酮能够促进SD大鼠成骨细胞生长和增殖,浓度适当时与雌二醇无显著差别,最佳浓度为1×(10-7~10-9)mmol/L,其作用可能与刺激骨保护素因子有关。当浓度升高至1×10-5mmol/L时,并不利于成骨细胞的增殖。更多还原

【Abstract】 BACKGROUND: Dehydroepiandrosterone (DHEA) derived from adrenal cortex has an indirect bioeffect through differentiating into androgen or estrogen in the related peripheral tissues. OBJECTIVE: To verify the effects and mechanisms of DHEA on promoting osteoblasts (OBs) proliferation. DESIGN, TIME AND SETTING: The grouping controlled experiment was accomplished in Shanghai Sixth People’s Hospital affiliated to Shanghai Jiao Tong University and Institute of Radiation Medicine, Fudan University from January 2006 to February 2007. MATERIALS: Ten SD rats were recruited, 1 day old, regardless of gender. DHEA was the product of American Sigma Company. METHODS: Rat OBs were separated and cultured in vitro. First generation of OBs cultured in a varied concentrations of DHEA (1×10-5, 1×10-7 and 1×10-9 mmol/L) for 72 hours were set up as the experiment group, while those exposed to 1×10-8 mmol/L of estradiol culture were regarded as positive controls and blank control group was set up as well. MAIN OUTCOME MEASURES: The OBs were identified by cell morphology and alkaline phosphatase (ALP) dyeing. Light microscope and methyl thiazolyl tetrazolium assay were used to detect the growth and proliferation of OBs. The formation of mineralized nodus was examined by alizarin bordeaux stain. The concentrations of osteoprotegerin in DHEA culture solution were also measured by double antibody sandwich ABC-ELISA simultaneously. RESULTS: ①Identification of primarily cultured OBs showed ALP dyeing was positive.②Proliferation rate of OBs was increased in experiment groups compared with that of the blank control group, and the most significant increase occurred in the concentration of 1×10-7 mmol/L DHEA culture (P < 0.01). However, there was no significant difference in proliferation rate between estradiol culture and DHEA group (P > 0.05).③ALP activity in experiment groups was higher, especially in the group with the concentration of 1×10-7 mmol/L DHEA (P < 0.01). No significant differences were seen in the ALP activity between estradiol culture and DHEA groups with concentration of 1×10-7 and 1×10-9 mmol/L (P > 0.05).④ALP activity of unitary cell population was higher in 1×10-9 mmol/L DHEA culture group than that in blank control group (P < 0.05).⑤Both the area and area ratio of mineralized nodules were significantly increased in various concentrations of DHEA groups (P < 0.01), which was similar to that of estradiol culture.⑥Responds of osteoprotegerin to DHEA were the most active in 1×10-9 mmol/L DHEA group after 72 hours of the culture (P < 0.01), and had a positive correlation with OBs proliferation. CONCLUSION: DHEA may promote growth and proliferation of rat OBs, its effect at the optimal concentration of 1×(10-7- 10-9) mmol/L is identical with estradiol, and might be related to stimulating osteoprotegerin pathway DHEA at the concentration exceeding 1×10-5 mmol/L has no effect on OBs proliferation.更多还原