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目的:配制缓释BrdU,用于检测前腭缝牵张成骨过程中成骨细胞的增殖和分化,探讨前腭缝牵张成骨的机制。方法:用泊洛沙姆温敏凝胶配制缓释BrdU,大鼠皮下注射后不同时间点采取血样,高效液相色谱法测定BrdU血药浓度,并与腹腔注射BrdU溶液进行比较。选取35天龄雄性SD大鼠40只,随机分为实验组和对照组,实验组安放扩大簧牵张前腭缝。2组动物于24h后随机皮下注射BrdU温敏凝胶1次或腹腔注射BrdU溶液1次。所有动物分别于48h和96h处死(n=5),免疫组化染色检测细胞的增殖和迁移。结果:皮下注射BrdU温敏凝胶6h后,血清中仍可检测出1.21μg/mL BrdU,免疫组化染色可见大鼠前腭缝有BrdU阳性的增殖细胞。腹腔注射BrdU溶液3h后,血清中仅检测到0.17μg/mL BrdU,免疫组化染色未见BrdU阳性细胞。前腭缝牵张48h后,骨缝边缘可见大量BrdU阳性成骨细胞;96h后可见部分BrdU阳性细胞包埋于骨缝边缘的新生骨基质中。结论:BrdU温敏凝胶能起到良好的缓释效果,为体内标记成骨细胞提供了一种简便易行的方法。牵张力作用于前腭缝后,促进骨缝边缘的间充质细胞增殖,这些细胞分化为成骨细胞并分泌骨基质,从而促进新骨形成。
OBJECTIVE: To prepare sustained-release BrdU for the detection of proliferation and differentiation of osteoblasts during the process of distraction osteogenesis in the anterior palate sutures and to explore the mechanism of distraction osteogenesis in the anterior palate. Methods: Sustained-release BrdU was prepared with poloxamer thermosensitive gel. Blood samples were taken at different time points after subcutaneous injection in rats. BrdU concentration was determined by HPLC, and compared with BrdU solution injected intraperitoneally. Forty rats of 35-day-old male SD rats were randomly divided into experimental group and control group. Two groups of animals were randomly injected subcutaneously with BrdU thermo-sensitive gel 1 hour or intraperitoneally with BrdU solution once a day. All animals were sacrificed at 48h and 96h (n = 5), and the proliferation and migration of cells were detected by immunohistochemistry. Results: After BrdU thermosensitive gel was injected subcutaneously for 6 hours, 1.21μg / mL BrdU could still be detected in the serum. BrdU positive proliferating cells were observed in the anterior palatal suture by immunohistochemical staining. After intraperitoneal injection of BrdU solution for 3h, only 0.17μg / mL BrdU was detected in the serum and no BrdU positive cells were detected by immunohistochemistry. A large number of BrdU-positive osteoblasts were seen at the edge of the seams after 48 h of anterior palate suture. After 96 h, some BrdU positive cells were embedded in the new bone matrix at the edge of the suture. CONCLUSION: BrdU thermosensitive gel has a good sustained-release effect and provides an easy and convenient method for labeling osteoblasts in vivo. Stretching affects the anterior palate seams, promoting the proliferation of mesenchymal cells at the edge of the seams, which differentiate into osteoblasts and secrete bone matrix to promote new bone formation.