摘要
目的 用酶消化法从正常人松质骨中分离成骨细胞 ,进行体外培养和功能鉴定 ,观察用 1 7β 雌二醇 (1 7β E2 )和雌激素受体拮抗剂ICI1 82 780处理后 ,成骨细胞中护骨素 (OPG)的表达变化。方法 用胰酶 -胶原酶消化法从正常人松质骨中分离培养成骨细胞 ,I型胶原染色用VanGie son法 ,钙化结节用茜素红染色 ,用钙钴法显示成骨细胞中碱性磷酸酶的表达 ,骨钙素和OPG基因的表达用RT -PCR检测 ,OPG蛋白用WesternBlot检测。结果 用酶消化法分离的人成骨细胞 ,在体外培养时可维持合成I型胶原、碱性磷酸酶、骨钙素 ,并形成钙化结节等功能 ;体外培养的人成骨细胞表达OPG ,1 7β E2 上调其表达 ,并能被雌激素受体拮抗剂ICI1 82 780阻断。结论 用酶消化法进行人成骨细胞培养 ,方便易行 ,可培养大量高纯度人成骨细胞供研究使用 ;1 7β E2 上调OPG表达 ,而绝经后雌激素缺乏时 ,OPG的表达相应减少 ,可能是骨质疏松的发病因素之一。
Objective\ To culture and identify the functions of human osteoblasts(HOB) in vitro ,the cells were harvested from the trabecular bone of normal subjects.Then the expression of osteoprotegerin(OPG)in the HOB cells treated with 17β estradiol and antiestrogen reagent ICI182780 was investigated. Methods\ The HOBs were isolated from the trabecular bone by trypsin and collagenase digestion.Type I collagen,mineralized nodes and the expression of alkaline phosphatase were studied by van Gieson's staining,alizarin red staining and modified Gomori method respectively.The expression of osteocalcin and OPG mRNA were examined by semiquantitive RT-PCR and OPG protein by Western blot. Results\ The HOBs purified by enzyme digestion in vitro functioned normally as observed in vivo ;OPG was expressed by the HOBs and could be upregulated by 17β estradiol,and this effect could be blocked by ICI182780,an antagonist of estrogen receptors. Conclusion\ It is easy to purify plentiful HOBs with normal functions by enzyme digestion,and the expression of OPG in HOBs is down regulated under the condition of estrogen deficiency.\;
出处
《中国骨质疏松杂志》
CAS
CSCD
2002年第4期283-286,共4页
Chinese Journal of Osteoporosis
