The Molecular Mechanism Of CPT-induced Apoptosis In Human Peripheral Blood Source Osteoclasts

Objectives:Tumor necrosis factor-related apoptosis-inducing ligand(Tumor Necrosis factor-related apoptosis- inducing ligand, TRAIL), a new member of tumor necrosis factor(Tumor Necrosis factor, TNF)superfamily.Which can induce a variety of tissue-derived tumor cell apoptosis in vitro, but almost have no effect on normal cells, the effect of TRAIL on osteoclast researches are rare reproted.Our research confirmed the cyclization allosteric tumor necrosis factor-related apoptosis-inducing ligand(Circular Permuted TRAIL, CPT; Beijing East Sha Biotechnology Co., Ltd. R&D) can be more effectively than the TRAIL on apoptosis. We studied the effect of CPT on osteoclasts induced from PBMC, analysis the m RNA expression levels of two CPT death receptors and two types of decoy receptors after the treatment of chemotherapy drugs, and explored the mechanism of the CPT on osteoclasts,propose a new treatment method and theoretical basis for the treatment of myeloma bone disease and so on.Methods:1 Culture of osteoclasts induced from peripheral blood cell Take the healthy human peripheral blood 10 ml, aseptic isolate PBMC by lymphocyte separation medium, seeded in 24-well platesin of different cell density(5×10~5,1×10~6?2×10~6cells/well) containing 30 ng/ml M-CSF and 30 ng/ml RANKL,culture for 21 days,form the giant,shape irregular andmulti nuclear(three or more nuclei) cell.2 Identification of osteoclasts2.1 Tartrate-resistant acid phosphatase(tartrate resistant acid phosphatase, TRAP) staining, multinuclear(three or more nuclei),the cell body is bigger, mature osteoclasts is wine red. With a microscope(100×) observed and photographed under ordinary light. Each taking four holes counted in 10 randomly selected three or more nuclei of osteoclasts.2.2 Scanning electron microscope to observe osteoclasts in bovine cortical bone formation after bone absorption in lacunae.3 CPT-induced apoptosis of osteoclasts3.1 Different concentrations of CPT effect on osteoclast induced apoptosis, CPT drug concentration respectively 100 ng/ml, 500ng/ml, role in 24 hours, 48 hours after the TRAP staining, observed the osteoclast morphological changes under inverted microscope.4 CPT receptor mRNAIn 0ng / ml, 100 ng / ml, 500 ng / ml CPT treated osteoclasts 24 h, 48 h, the real-time quantitative PCR(realtime fluorescence quantitative PCR,RT-PCR) assay DR4, DR5, Dc R1 and Dc R2 m RNA expression levels, to analyze its relevance to the osteoclast apoptosis induced by CPT.Results:1 The peripheral blood mononuclear cells induced by M-CSF and RANKL for 21 days,then a large number of mature osteoclasts can be observed by the microscope, the morphological features are large cell volume, rich in wine red cell plasma, multinuclear, nucleus?3 or more higher. TRAP staining indicted the cell were TRAP positive(red wine),bone absorption experiments showed bone resorption activity of the cell.The result demostrate that PBMC can induced to osteoclast. Under the same culture conditions PBMC cells at 2 × 10~6cells/ml inoculum density, induce the formation of osteoclast number was 50.2 ± 11.6/ HPF,at 5 × 10~5cells/ml induced by the number of osteoclast formation of 18.0± 6.37 / HPF and 1 × 10~6cells/ml induced the formation of osteoclast number as compared to 36.4 ± 9.54/ HPF,so the 2 × 10~6cells/ml induce the largest formation number of osteoclasts.2 CPT can induce apoptosis of osteoclasts, the CPT observed under the microscope after 24 hours of osteoclast vacuolar, deformation, rupture or disappearance of features of apoptosis 48 h after osteoclast closure tape appears.3 The Annexin V-FITC fluorescence dyeing kit, fluorescence microscopy to CPT acting on the osteoclasts typical early apoptotic features,inducing osteoclast apoptosis showed a certain period of time- concentration effect.4 RT-PCR to detect the expression of osteoclast-associated receptor in m RNA level of CPT,DR4, DR5, Dc R1 and Dc R2 on osteoclasts were expressed. After 0 ng/ml, 100 ng/ml, 500 ng/ml CPT processing osteoclast 24 h, CPT related receptors DR4, DR5 and Dc R1 and Dc R2 m RNA expression level change was not significant(P>0.05);post-processing 48 h, DR4 and Dc R2 the m RNA expression levels still no significant change(P> 0.05), m RNA expression levels of DR5 and Dc R1downregulation(P<0.05 or P<0.01). CPT is inferred by DR5-induced apoptosis of OC.Cnclusions:1 Human osteoclast which have bone resorption activity can be induced from PBMC in vitro.2 CPT can induce the apoptosis of human osteoclast which induced from peripheral blood in a time and concentration-dependent manner.3 CPT can reduce the osteoclast mRNA expression level of DR5 and Dc R1 but does not affect DR4 and Dc R2 in the indicated time, concluded that CPT apoptosis by DR5, Dc R1 involved in the regulation of OC.