低氧对体外共培育体系破骨细胞分化影响的试验研究-第三军医大学学报.docVIP

不同氧浓度对小鼠骨髓细胞形成破骨样细胞影响的实验研究 郎红梅　　金小岚※ 万勇 游志清 （成都军区总医院内分泌科，四川，成都，610083） 【摘要】目的：通过观察不同氧浓度对小鼠骨髓细胞形成破骨样细胞的影响，探讨低氧对破骨细胞生成影响的分子机制。方法：取6～9周龄KM小鼠骨髓细胞，用含1,25(OH)2D3(10-8mol/L)、地塞米松(10-8mol/L) 和 10％胎牛血清的α－MEM培养基分别在氧浓度为20％（常氧组）和6％、3％，1％（低氧组）中培养。骨髓细胞纯化后加入到第3~5代成骨细胞进行共培养，随机分组，每组样本数为4，选择缺氧第5天进行下面的实验，每项试验重复3次。用TRIzol提取细胞总RNA，用半定量逆转录PCR(RT-PCR)检测OPG、RANK及RANKL mRNA的表达；用Western blotting检测RANK蛋白的表达情况。结果：与常氧组相比，OPGmRNA的表达随着氧浓度的降低而降低，RANKmRNA和RANK蛋白的表达随着氧浓度的降低而升高，RANKLmRNA的表达随着氧浓度的降低而升高。结论：低氧可促进破骨样细胞的生成，尤其在3％氧浓度时，破骨细胞生成达最高。 【关键词】 低氧；破骨细胞 ；OPG ； RANKL ；RANK Experimental Study of the Effects of Different Oxygen Concentration on the Formation of Mouse Osteoclastic Cells in Vitro Jin Xiao-lan,Lang Hong-mei,Wan yong et al.Department of Endocrinology,Chengdu Military Command General Hospital, Chengdu,610083,China 【Abstract】Objective To investigate if the formation of mouse osteoclastic cells is affected by hypoxia . Methods Mouse bone marrow cells were obtained from the tibiae of six-to-nine-week-old male mice, the cells were cultured in α－MEM culture medium containing 1α,25(OH)2D3(10-8mol/L) ,Dexamethasone(10-8mol/L) and 10％ fetal bovine serum. After the bone marrow cells were purified, the 3-5th generation osteoblast were added to .Co-incubation cells were divided into groups at random, each group has 4 samples, and then the cells were cultured for five days in normoxia(20％O2) and hypoxia(6％O2、3％O2、1％O2 ) respectively. The following experiments were done in fifth day and each experiment was repeated three times: analysing the expression of OPG, RANK and RANKLmRNA by the method of reverse transcription polymerase chain reaction(RT-PCR),detecting expression of RANK protein by the method of western blotting. Results Oxygen concentration have significant effect on the formation of osteoclastic cells. With decrease of oxygen contents, compared with normoxia group,the expression of OPGmRNA in hypoxia groups decreased markedly; the expression of RANKmRNA and RANK protein increased;th