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目的:观察益骨胶囊含药血清对体外培养的大鼠成骨细胞骨保护蛋白mRNA的影响。方法:实验于2004-05/2005-02在广州暨南大学生命科学技术学院细胞与蛋白质工程实验室完成。选取12月龄SD雌性大鼠20只,随机分为2组。中药给药组(益骨胶囊由淫羊藿、当归、白芍、枸杞子等8味中药组成)和蒸馏水对照组,每组10只。运用大鼠灌胃的方法制备含药血清和对照血清,分离、培养新生大鼠的颅骨成骨细胞,传代后分为2组(含药血清组和空白血清对照组),每组8孔,分别在培养24,48h时用反转录聚合酶链反应技术测定特异性电泳条带所显示成骨细胞中骨保护蛋白mRNA的相对表达量,酶联免疫法检测48,72和96h时200mL/L终浓度培养上清中骨保护蛋白的浓度。结果:两组动物各10只,均无亡失,所取血清正常用于细胞培养。①成骨细胞在接种4h后可见部分细胞贴壁、伸展。随培养时间延长,细胞伸出较多突起,细胞形态为单核,多边形及梭形,培养2d细胞基本全部贴壁,主要为梭形或多边形,培养6d左右基本贴满瓶壁,呈铺路石样排列。单层铺满培养瓶后可出现重叠生长,传代细胞大约15d天左右开始形成矿化结节。②特异性电泳条带显示益骨胶囊含药血清培养后24h和48h成骨细胞骨保护蛋白mRNA的表达均高于空白血清对照组犤(0.196±0.015,0.236±0.016),(0.152±0.018,0.206±0.022),P<0.01犦;③200mL/L终浓度的益骨胶囊含药血清培养后48,72和96h成骨细胞骨保护蛋白的表达明显高于空白血清对照组犤(0.1230±0.0128,0.1681±0.0130,0.1983±0.0186),(0.1067±0.0106,0.1385±0.0126,0.1588±0.0121),P<0.01或P<0.05犦。结论:益骨胶囊含药血清具有促进成骨细胞表达骨保护蛋白的作用。提示益骨胶囊能够通过对成骨细胞的影响而对骨吸收环节具有抑制作用,但是否对其他信号通路也有作用尚待研究。
Objective: To observe the effect of Yigu capsule drug-containing serum on osteoprotegerin protection protein mRNA in vitro. METHODS: The experiment was performed at the Cell and Protein Engineering Laboratory, School of Life Science and Technology, Jinan University from May 2004 to February 2005. Twenty female SD rats aged 12 months were randomly divided into 2 groups. The traditional Chinese medicine administration group (Yigu Capsule consists of 8 Chinese herbal medicines, Epimedium, Angelica sinensis, Radix Paeoniae Alba, Gardenia jasminoides, etc.) and distilled water control group, 10 in each group. The rat serum containing the drug and the control serum were prepared by intragastric administration. The osteoblasts of newborn rats were isolated and cultured. After passage, they were divided into 2 groups (containing drug serum group and blank serum control group) with 8 holes in each group. The relative expression levels of osteoprotegerin mRNA in osteoblasts were determined by reverse transcription-polymerase chain reaction (PCR) at 24 and 48 hours of culture, and 200 mL/hr at 48, 72 and 96 h of enzyme-linked immunosorbent assay. The concentration of bone protective protein in the culture supernatant of L final concentration. RESULTS: Ten animals in each group were absent. The serum was used for cell culture. 1 After osteoblasts were inoculated 4 h, some cells adhered and stretched. With the extension of culture time, the cells protruded more protuberances. The cell morphology was mononuclear, polygonal and fusiform. The 2d cultured cells were almost all adherent, mainly fusiform or polygonal, and the bottle was plastered about 6 days after the culture. Sample arrangement. Overlapping growth can occur after a single-layered confluent culture flask, and mineralized nodules begin to form around 15 days in the passaged cells. 2 Specific electrophoresis bands showed that the bone protective protein mRNA expression of osteoblasts at 24h and 48h after culture with Yigu capsule contained serum was higher than that of blank serum control group (0.196±0.015, 0.236±0.016), (0.152±0.018). 0.206±0.022), P<0.01犦; bone marrow capsule osteoblast protein expression at 48, 72 and 96 h after culture with 3200 mL/L final concentration of Yigu capsule contained serum was significantly higher than that of blank serum control group (0.1230±0.0128, 0.1681 ± 0.0130, 0.1983 ± 0.0186), (0.1067 ± 0.0106, 0.1385 ± 0.0126, 0.1588 ± 0.0121), P <0.01 or P <0.05 犦. Conclusion: Yigu capsule drug-containing serum can promote the expression of osteoprotegerin in osteoblasts. It is suggested that the benefit bone capsule can inhibit bone resorption by affecting the osteoblasts, but whether it has effects on other signaling pathways still needs to be studied.