The Mechanism Of 17β-Estrodiol On The Activity Of Human Osteoblast-like Cells And Osteoclast-like Cells

Postmenopausal osteoporosis is the most common type of primary osteoporosis. There is an accelerated phase of bone loss in the earlier period of menopause corresponding to the decrease of the 17 P -estrodiol (17 -E2) level. Many researchers showed that in the osteoblast-like cells and osteoclast-like cells (OCLs) 17 -E2 can alter the expression of pro inflammatory factors, including OPG, RANKL, ILH5 and IL-11 etc. In this research the multinuclear giant cells with the phenotypes of mature osteoclasts were isolated and purified from human giant cell tumour of bone. After treated with 17 -E2 the expression of receptor activator of nuclear factor-kappaB (RANK) , cathepsin K (CK) and calcitonin receptor (CTR) mRNA was detected. In order to study the function of osteoblasts mediated by 17 -E2, MG63 cells were treated with 17 -E2, then the expression of IL-6, IL-11 and NF- K B were detected by RT-PCR, Northern blot and Western blot respectively. The result showed: (1) human giant cell tumour of bone is an appropriate source of OCLs, from which a lot of biologically active OCLs can be isolated, and OCLs are very suitable for study on osteoclasts in vitro. (2) 17 -E2 can down-regulate the expression of CK mRNA in OCLs, but has no effect on the expression of RANK or CTR mRNA. (3)17 -E2 can down-regulate the expression of IL-6 and IL-11 mRNA in MG63 cells. (4) NF- K Bis the important signaling factor through which 17 -E2 mediates the expression of IL-6, and it can be down-regulated by 17 -E2 in MG63 cells.Part One A new method to isolate large numbers of human osteoclast-like cells in VitroObjective: To purify and identify the osteoclasts-like cells (OCLs) from human giant cell tumor of bone(GCT).Method: A new method was developed that allows the purification oflarge numbers of OCLs. The OCLs were isolated from GCT with 0.25% trypsin, 0. 2% collagenase and 25 m filtration web, and characterized in terms of the expression of different phenotypic markers of osteoclasts (OCs) and their ability to resorb bone matrix. The phenotypic markers of DCs include the expression of tartrate-resistant acid phosphatase (TRAP), acid phosphatase (ACP), resorption pit assay, reaction to calcitonin and identification of F-actin. The expression of calcitonin receptor(CTR), cathepsin K(CK) and receptor activator of necrosis factor B (RANK) mRNA were examined by RT-PCR.Result: The OCLs purified by the established techniques functionednormally in vitro, with a yield of about 80. 1%, and the ability to express CTR, CK and RANK mRNA.Conclusion: The established new techniques for purifying OCLs could provides enough amount of OCLs for study.