摘要
目的 获得足量高纯度活性破骨细胞进行破骨细胞生化学和分子生物学研究。方法利用破骨细胞与Ⅰ型胶原基质的高亲合力 ,先用pronase和EDTA作用除去未贴附的造血细胞及结合较疏松的间质细胞 ,继而用 0 0 1%胶原酶作用进一步除去残留的间质细胞 ,最后用 0 .1%胶原酶作用得到高纯度破骨细胞悬液。分离细胞用抗酒石酸酸性磷酸酶 (TRAP)染色 ,荧光标记显示细胞骨架 ,RT PCR方法检测破骨细胞标志酶基因。结果 胶原基质培养结合酶消化可大大提高破骨细胞纯度 ,破骨细胞对降钙素反应 ,并表达MT1 MMP ,MMP 9,组织蛋白酶K和TRAP基因mRNA ,可产生吸收陷窝。结论 利用兔破骨细胞与Ⅰ型胶原的亲合力 ,可获得多量高纯度破骨细胞 ;并表达特征性基因。
Objective\ To obtain enough amount of viable,highly enriched osteoclasts for the biochemical and molecular biological research of osteoclasts. Methods\ Rabbit osteoclasts were isolated by the modified methods of Kakudo et al.in which the high affinity of osteoclasts with collagen type Ⅰ was used,the non osteoclast cells were renoved by enzymatic treatment.The morphology obsevation,TRAP staining,calcitonin reaction and resorpion lacunae of obtained osteoclastss were conducted.The gene expression of some osteclast marker enzymes was examined by RT PCR. Results\ The typical characteristics of osteoclasts were demonstrated and the transcriptional expression of MT1 MMP,MMP 9 and cathepsin K in osteoclasts was also detected. Conclusion\ A large amount of highly enriched osteoclasts can be harvested by utilizing the affinity of rabbit osteoclasts with collagen type Ⅰ.At least,at transcriptional expression level,the highly enriched osteoclasts express MT1 MMP,MMP 9 and cathepsin K.\;
出处
《中国骨质疏松杂志》
CAS
CSCD
2001年第1期15-17,共3页
Chinese Journal of Osteoporosis
关键词
破骨细胞
纯化
基因表达
细胞培养
Osteoclasts
\ Culture
\ Purification
\ Gene expressiH
