大鼠听泡原代成骨细胞分离培养及鉴定  

The Primary Osteoblasts Isolation,Culturing and Identification in Rat Otocyst

作　　者：黄玉 刘豆 黄文霞 梁耕田[1] Huang Yu;Liu Dou;Huang Wenxia;Liang Gentian(Department of Otolaryngology-Head and Neck Surgery,the Third Hospital of Wuhan,Wuhan,430060,China)

机构地区：[1]武汉大学附属同仁医院(武汉市第三医院)耳鼻咽喉-头颈外科,武汉430060

出　　处：《听力学及言语疾病杂志》2023年第5期439-443,共5页Journal of Audiology and Speech Pathology

基　　金：武汉市科技局知识创新专项曙光计划(2022020801020554);武汉市卫健委青年项目(WX21Q24);武汉市卫健委面上项目(WX21A10);武汉市卫健委面上项目(WX18C05)。

摘　　要：目的对SD大鼠听泡的原代成骨细胞进行分离培养及鉴定。方法选取6只出生24 h的SD乳鼠,从颞区无菌分离出听泡组织,经0.25%胰蛋白酶和I型胶原酶消化后接种培养;待生长融合至80%密度时,进行传代培养,于倒置显微镜下观察原代及传代成骨细胞的生长状态,并用EDU法分析其增殖趋势。对生长良好的细胞进行成骨诱导分化,并用ALP试剂盒和茜素红试剂盒进行染色分析,鉴定分化效果。结果大鼠听泡原代及传代成骨细胞培养中,细胞均贴壁良好、生长旺盛,第2 d分别可融合生长至50%、30%的密度,第4 d分别可融合生长至80%、70%的密度,形态呈现多样化。EDU法检测分析见:第1~4 d细胞增殖比均维持在较高水平,从第5 d起呈直线下降趋势。经诱导分化后,细胞生长状态良好,形态依旧多样化,早期以梭形为主,中后期形态较为丰腴。分化后2周,ALP染色见大量细胞胞质呈现深蓝色;第15 d经茜素红染色便可见局部出现橙红色钙化结节。结论通过胰酶联合胶原酶两步消化法,成功地从SD乳鼠听泡中分离出原代成骨细胞,所得细胞生长及传代能力强,经诱导分化后具备良好的成骨活性,为鼓室硬化等疾病的体外研究提供可能。Objective To isolate,culture and identify the primary osteoblasts in SD rat otocyst.Methods The otocyst was isolated aseptically from temporal region of six SD rats which were born in 24 hours.After digestion with 0.25%trypsin and type I collagenase,the primary osteoblasts had been inoculated and cultured.When the cells grew and fused to 80%density,they were subcultured.The growth status of primary and subcultured osteoblasts was observed under inverted phase contrast microscope,and the proliferation trend was analyzed by EDU method.The cells with good growth were osteogenic induced and differentiated.ALP kit and alizarin red kit were used for identification of differentiation effect.Results In the primary culture and subcultured osteoblasts of rat otocyst,the cells were well adherent and grew vigorously.The cells could fuse and grow to 50%and 30%density on the second day,and 80%and 70%density on the fourth day,respectively,with diversified morphology.It showed that the cell proliferation ratio maintained at a high level from day 1 to day 4,and a straight downward trend from day 5 by EDU test.After induced differentiation,the cells grew well and diversified in morphology.In the early stage,the cells were mainly spindle shaped,and in the middle and late stage,the cells were plump.At 2 weeks after differentiation,a large number of cells showed dark blue cytoplasm by ALP staining.On the 15th day,local orange-red calcified nodules were observed by alizarin red staining.Conclusion The primary osteoblasts were successfully isolated from the otocyst of SD rats by the two-step digestion method of trypsin and collagenase.The obtained cells had strong growth and passage ability,and had good osteogenic activity after induction and differentiation,which laid a foundation for the study of tympanosclerosis and other diseases in vitro.

关 键 词：大鼠 听泡 成骨细胞 鼓室硬化 

分 类 号：R764[医药卫生—耳鼻咽喉科]