中文摘要：

目的：构建可稳定表达成纤维细胞生长因子18蛋白的真核表达载体，观察成纤维细胞生长因子18对体外培养的兔骨髓基质干细胞碱性磷酸酶合成的影响。 方法：本实验于2002-10／2004-03在解放军第四军医大学全军骨肿瘤研究所及121腔医学院完成。①将pGEM-T-FGF18-3和pSecTag2B表达载体分别用KpnⅠ和NotⅠ酶切后重组pSecTag2B-FGF18质粒；ELISA检测转染后COS-7细胞上清的成纤维细胞生长因子18蛋白，以大于阴性对照A值2.1倍以上为阳性结果。②用细胞上清刺激骨髓基质干细胞，根据含小牛血清的DMEM不同比例稀释转染后COS-7细胞上清分为1：2稀释组、1：4稀释组、1：8稀释组、1：16组、1：32稀释组；对照组：用含体积分数为0．02小牛血清的DMEM培养液按1：2稀释未转染的COS-7细胞上清。⑧通过酶动力法检测细胞碱性磷酸酶合成情况。 结果：①成功构建了pSecTag2B-FGFl8真核表达载体。②转染哺乳动物细胞COS-7用夹心ELISA法检测到了成纤维细胞生长因子18蛋白质的稳定表达，原液及l：10稀释度细胞上清A值大于阴性对照A值2．1倍以上（P〈0．01）。③用细胞上清刺激骨髓基质干细胞72h后，1：2稀释组、1：4稀释组、1：8稀释组、1：16组、1：32稀释组骨髓基质干细胞A值较对照组明显增高（P〈0．01），证实了成纤维细胞生长因子18对体外培养的兔骨髓基质干细胞碱性磷酸酶合成的促进作用。 结论：成纤维细胞生长因子18有显著促进体外培养的兔骨髓基质干细胞碱性磷酸酶合成的作用，提示成纤维细胞生长因子18对于骨髓基质干细胞的分化及成骨表型的维持起着重要的调节作用。

英文摘要：

AIM： To construct stable eukaryotic expression vector of fibroblast growth factor （FGF） 18 and observe the effects of FGF-18 on synthesis of alkaline phosphatase in bone marrow stem cells （BMSCs） of rabbits cultured in vitro. METHODS： The experiment was performed in the Orthopedics Oncology Institute of Chinese PLA and Dental Hospital, Fourth Military Medical University of Chinese PLA from October 2002 to March 2004.①Expression vector of pGEM-T-FGF18-3 and pSecTag2B were reconstructed into pSecTag2B-FGF18 plasmid with Kpn Ⅰ and Not Ⅰ enzyme; Protein of FGF- 18 in cellular supernatant of transfected COS-7 was detected with ELISA, those above 2.1 times of A value in negative control were positive results.②Cellular supernatant was used to stimulate BMSCs. Cellular supernatant of transfected COS-7 were divided into 1：2 diluting group, 1：4 diluting group, 1：8 diluting group, 1：16 diluting group, 1：32 diluting group according to different proportions of DMEM containing calf serum （CS）; Control group： Cellular supernatant of untransfected COS-7 was diluted with DMEM culture fluid, in which the volume fraction of CS was 0.02, according to 1：2.③Synthesis of alkaline phosphatase in cells was inspected with enzyme kinetic method. RESULTS： ① Eukaryotic expression vector pSecTag2B-FGF18 was constructed successfully. ②Stable expression of FGF-18 in transfected mammalian cells of COS-7 was inspected with ELISA method. A value of stock solution and cellular supernatant with the dilution of 1：10 were 2.1 times than that in negative control （P 〈 0.01）. ③72 hours after the stimulation of BMSCs with supernatant, A values of BMSC in 1：4 diluting group, 1：8 diluting group, 1：16 diluting group, 1：32 diluting group were obviously increased than that of control group （P 〈 0.01）, which proved that there was promotion of FGF-18 on the synthesis of alkaline phosphatase in BMSCs of rabbits cultured in vitro. CONCLUSION： FGF-18 can remarkably promote synthesis of al