成纤维细胞生长因子Ⅱ型受体基因突变对小鼠骨髓基质细胞发育的影响

Fibroblast growth factor type Ⅱ receptor mutation affects the development of bone marrow stromal cells in mice

目的:观察对比野生型小鼠和成纤维细胞生长因子Ⅱ型受体突变型小鼠骨髓基质细胞体外培养的生长情况以及成骨潜能,探讨成纤维细胞生长因子Ⅱ型受体功能持续增强对小鼠骨髓基质细胞发育的影响。方法:实验于2005-09/2006-02在解放军第三军医大学附属大坪医院野战外科研究所创伤中心实验室,创伤、烧伤与复合伤国家重点实验室完成。①实验动物:将雄性成纤维细胞生长因子Ⅱ型受体S252W突变小鼠(由陈林教授在美国国立卫生研究院建立后引入本实验室)和雌性C57BL/6J小鼠(由解放军第三军医大学大坪医院实验动物中心提供)交配,经过基因型鉴定的雄性F1代8周龄小鼠按照基因型分为野生型组和突变型组,12只/组。②实验方法:将两组小鼠脱臼处死后分离胫、股骨,用DMEM培养液冲出骨髓,离心后弃上清,加入DMEM低糖培养液制成细胞悬液,贴壁法分离培养骨髓基质细胞,以1×108 L-1密度传代。③实验评估:倒置相差显微镜下观察细胞形态及贴壁生长情况。采用MTT法检测两组骨髓基质细胞的增殖情况。检测体外成骨诱导条件下两组骨髓基质细胞的碱性磷酸酶活性。采用Vankossa染色检测体外成骨诱导条件下两组骨髓基质细胞的钙结节形成能力。结果:①形态学观察:倒置相差显微镜下两组骨髓基质细胞形态变化相似。第3代骨髓基质细胞在体外诱导成骨条件下增殖速度明显减慢,多数细胞由梭形变成长方形,并逐渐成为立方形,细胞体积增大。②生长曲线:野生型组骨髓基质细胞的吸光度值在接种后2d内变化不大,细胞处于潜伏期,第4天显著增加,第6天达高峰,进入平台期。与野生型组比较,突变型组骨髓基质细胞生长曲线出现右移和平台期滞后。③碱性磷酸酶活性:在体外诱导成骨条件下,野生型组骨髓基质细胞随培养时间延长碱性磷酸酶活性逐渐上升,第9～15天达高峰,然后随细胞老化而下降。与野生型组相比较,突变型组骨髓基质细胞碱性磷酸酶活性增高出现滞后现象,并且其高峰数值显著低于野生型组(P<0.05)。④钙结节形成:经过21d的成骨诱导培养,野生型组骨髓基质细胞可见较多的钙结节形成,突变型组有分散的钙结节形成,但较细小。结论:成纤维细胞生长因子及其受体信号对细胞发育的调控效应是复杂多面的,持续的成纤维细胞生长因子Ⅱ型受体功能增强可能引起体外培养的小鼠骨髓基质细胞增生减缓,并可能阻抑其向成骨细胞的分化。

AIM： To study the proliferation and osteogenic ability of bone marrow stromal cells （BMSCs） of wild type mice and fibroblast growth factor type Ⅱ receptor （Fgfr2） and to explore the influence of persistent enhanced Fgfr2 function on development of mice BMSCs. METHODS： The experiment was carried out at Laboratory of Trauma Center, Research Institute of Surgery, Daping Hospital, Third Military Medical University of Chinese PLA, State Key Laboratory of Trauma, Burn and Combined Injury from September 2005 to February 2006. ①Female mice with gain-of-function mutation of Fgfr2 （lead in this laboratory after establishing at National Institutes of Health by Professor Chen） were mated with male C57BL/6J mice （provided by Experimental Animal Center, Daping Hospital, Third Military Medical University of Chinese PLA）. Their F1 pups of mice aged 8 weeks were identified by PCR genotyping, which produced two fragments from mutant type （mutant type group, n =12） and one fragment from wild type （wild type group, n =12）. ②Mice of the two groups were sacrificed, and then anticnemion and femur were isolated to collect bone marrow with DMEM culture solution. After centrifuging, supernatant was removed and DMEM culture solution with low carbohydrates was added to make into cell suspension. BMSCs were harvested by attachment method and went down to passage at the density of 1×10^8 L^-1. ③Cell morphology and adherent growth were observed under inverted phase contrast microscope. The proliferation of BMSCs, alkaline phosphatase （AKP） activity of BMSCs cultured in osteogenic differentiation medium and mineral deposition in the two groups were measured by MTT assay, an AKP kit and Von Kossa staining, respectively. RESULTS： ①The morphological change of BMSCs from wild type mice was similar to that from mutant type mice. Proliferation of 3-generation BMSCs was very slow in osteogenic differentiation medium. Most cells changed from fusiform shape to oblong, gradually cube, and cell volume became larger. ②Cell growth curves： Absorbance （A） did not change greatly in BMSCs of the wild type group within 2 days after passage in logarithmic phase, gradually increased at day 4 and reached the highest A value at day 6, entered platform phase. Mutant type BMSCs entered platform phase later and the growth curve of mutant BMSCs showed a right-shift compared with that of wild type BMSCs. ③AKP activity of BMSCs： After been cultured in osteogenic differentiation medium, AKP activity increased in wild type BMSCs with the prolongation of culture time, and reached the peak at days 9-15, and then decreased with the cell senescence. The highest AKP activity in mutant type BMSCs appeared significantly later and the highest level was lower obviously than in wild type BMSCs （P 〈 0.05）. ④Calcium nodules was strong positive in wild type BMSCs but only showed weak staining in mutant type BMSCs at day 21 of the osteogenic culture. CONCLUSION： Fgfr2/Fgfr2 signaling has complex and diverse effects on cells development. Continued enhanced Fgfr2 function may inhibit the proliferation of BMSCs and suppress their osteogenic differentiation in vitro.