建立水合氯醛去除成骨细胞初级纤毛的细胞模型

Establishment of osteoblast primary cilla model removed by chloral hyrate

目的：建立水合氯醛去除成骨细胞初级纤毛的细胞模型,并观察去除成骨细胞初级纤毛对电磁场提高成骨细胞ALP染色和钙化结节染色的影响。方法：贴壁法分离培养3只出生3 d,体重6~9 g的雄性SD大鼠的乳鼠颅骨成骨细胞;待上述细胞生长至融合状态时传代培养并随机分成4组：不加水合氯醛组（对照组）、2 m M、4 m M和8 m M水合氯醛处理组;将上述4组细胞置于37℃、5%CO2的培养箱培养72 h,用激光共聚焦扫描显微镜观察初级纤毛形态,并用Image-Pro Plus 6.0软件分析成骨细胞初级纤毛发生率;筛选出能有效去除成骨细胞初级纤毛的水合氯醛浓度的细胞,并将其分为以下3组：正常对照组（control group,C）,电磁场处理（electromagnetic fields,EMFs）组及电磁场处理＋4 m M水合氯醛组。向上述3组细胞加入含10%FBS的DMEM培养液继续培养9 d,碱性磷酸酶组织化学染色观察ALP的形成;培养12 d,茜素红染色观察钙化结节的形成。结果：与对照组和2 m M水合氯醛组相比,4 m M水合氯醛能有效地去除成骨细胞初级纤毛的发生（P〈0.01）;去除成骨细胞初级纤毛消弱了EMFs促进成骨细胞ALP和钙化结节的形成,具体表现在：与EMFs组相比,EMFs＋水合氯醛组ALP和钙化结节的染色面积明显减少（P〈0.01）。结论：4 m M水合氯醛能有效去除成骨细胞的初级纤毛;初级纤毛部分参与了电磁场促进成骨细胞ALP和钙化结节的形成,为探讨电磁场促进成骨细胞成熟矿化的机理提供新思路。

Objective：To establish osteoblast model,primary cilla model was removed by chloral hyrate,observe effects of osteoblast primary cilla moved on enhancing ALP staining and calcified nodules staining in electromagnetic field. Methods：Three 3 day old male SD rats weighed between 6 and 9 g were killed,cranial osteoblast was drawed and adherencing cultured respectively. Cells were subcultured and randomly divided into 4 groups until reach to fusion states. The four groups included chloral hydrate non involved group（control group）,2 m M,4 m M and 8 m M chloral hydrate group,and cultured in 37 ℃,5%CO2incubator for 72 h. Morphology of primary cilla was observed by laser confocal scanning microscope,and incidence of osteoblast primary cilla was analyzed by Image-Pro Plus 6.0 software. Cells in the correct concentration group which can removed cillia most effectively were selected and divided into 3 groups,including control group（C）,Electromagnetic fields group（EMFs）,and EMFs with 4 m M chloral hydrate group. DMEM nutrient solution contained 10% FBS were added into three groups and cultured for 9 days and formation of ALP were observed by histochemical staining of alkaline phosphatase. After 12days＇ cultivation,formation of mineralization nodes was observed by alizarin red staining. Results：Compared with control group and 2m M chloral hydrate group,4 m M chloral hydrate group could effectively remove osteoblast primary cilla（P0.01）.Removal of osteoblast primary cilla could weaken the formation of ALP and mineralization nodes in osteoblast in EMFS. Compared with EMFs group,the area of ALP and mineralization nodes in EMFs with 4 m M chloral hydrate group were decreased obviously（P0.01）. Conclusion：4m M chloral hydrate could effectively remove osteoblast primary cilla. Primary cilla participate in EMFs promoting formation of ALP and mineralization nodes in osteoblast and provide new ideas for exploring mechanism of EMFs promoting osteoblast maturation and mineralization.