The Effect Of Signal-selective Parathyroid Horm One Analogs On The Gene Expression Profiles Of Mouse Osteoblast

BACKGROUNDOsteoporosis,presented by Pornmer in1885, is a gradual deepening with the historical development and technological progress.In the early time,it was generally believed that the bone loss of the whole body meant osteoporosis,but Americans thought that fractures of elder meant osteoporosis. Until the3rd and the Fourth International Osteoporosis Symposium held in Denmark in1990and in Hong Kong in1993respectively, osteoporosis had a clear definition which was accepted widely:Primary Osteoporosis, characterized by the loss of bone mass and bone degradation of microscopic structure which results in the increase of bone fragility and fracture,is a systemic skeletal disease.Every October20th of every year is difined’International Osteoporosis DayOsteoporosis epidemiological survey found that the prevalence of osteoporosis, over the age of40,60and80was16.1%,22.6%and50%recpectively with the increase of age. With the development of human society and the increase of medical standards and the level of healthy care, osteoporosis has gradualy become the focus of clinical care and treatment. China is gradually aging society, at the same time the social burden of osteoporosis gradually increased. The mechanism and treatment of osteoporosis has become a hot topic.The main treatment of osteoporosis is antiresorptive drugs,such as bisphosphonates, estrogen, calcitonin of denosumab (nuclear factor Kappa B ligand antibody), odanacatib (osteoclast enzyme of cathepsin K inhibitors), glucagon-like peptide-2, etc.These drugs mainly slow down the bone resorption, but the bone formation is limited. Bone resorption inhibitors decrease obviously the bone resorption and increase mildly bone formation,in that case, bone density increases. Antiresorptive drugs may produce some side effects, such as the difficulity of fracture heal because of combination of two kinds of sufficient bone resorption inhibitors.PTH(parathyroid hormone) is an important hormone which regulates bone metabolism in vivo, the only clinically available to promote bone anabolic drugs. Natural PTH has84amino acids. The the N-terminal34amino acid residues and84amino acids have the same funcation of receptor binding and activation.The study has confirmed that PTH(1-34) mainly activates PKA and PKC signaling pathways. With the research on the PTH signaling pathway in depth, the first two amino acids of PTH N-terminal play a important role in the PTH signal transduction. A serine (Ser) mutation to glycine (Gly) greatly weaken the PLC pathway activation of PTH.19glutamic acid mutation into arginine (Arg) can compensate for the weakening of receptor binding caused by the lack of29-34amino acid residues. Experimental results show that G1R19(1-28) and G1R19(1-34) active PLC pathway mildly, and G1R19(1-28) only actives cAMP/PKA signaling pathway.In addition to cAMP/PKA signaling pathway, G1R19(1-34) can active PKC (non-PLC/PKCO signal pathway due to the presence of PTH (29-34) structure. The three peptides can increase the bone mineral density of the distal femur in mice, and PTH (1-34) and G1r19(1-34) increase the amount of femoral bone mineral density more strongly than the G1R19(1-28).Wnt genes play an important role in embryonic development, adult tissue repair, and tumor occurrence, The research of Wnt signaling pathway for the regulatory role of the skeletal system, especially in osteoblasts and chondrocytes gradually increased recently. The Wnt signaling pathway plays an important role in regulating bone mass. The researchs show that PTH (1-84) can play osteogenesis by activating the Wnt signaling pathway. PTH plays osteogenesis through different signaling pathways, what kind of relationship exists between these pathways and the Wnt signaling pathway, still needs study in depth.OBJECTIVE1. In order to obtain a mild and efficient method of primary osteoblast culture.This study use type Ⅰ and type Ⅱ collagenase,instead of traditional method.2. To get the different genes among three PTH analogs by gene chip, explore the relationship between the PTH and Wnt signaling pathway, to provide the theoretical basis for the PTH’s bone formation effect.METHODS1. Osteoblasts were isolated from calvaria of2-day-old C57BL neonatal mouse and identified by observation using the inverted phase contrast microscope, ALP staining, and Alizarin red staining2. The cells at passage1with90%confluent were divided into four groups:control group, PTH (1-34) group, G1R19(1-34) group, G1R19(1-28) group. Then the medium was changed to α-MEM supplemented with1%FBS. After12h, vehicle or three peptides (10nM PTH (1-34)、10nM G1R19(1-34) and100nM G1R19(1-28)) were added into the culture medium.4h later, cells were washed gently with cold PBS three times before total RNA was isolated. The RNA is delivered to the company for the gene chip experiment. Then we will analysi the data to gei the Wnt-related factors changed.3. Expression of Wnt related genes was measured by Quantitative RT-PCR(three times repated). Data presented by X±S statistics done with SPSS13.0. One-way AN OVA were used among groups, LSD test were used between groups, P<0.05presents statistics different.RFSULTS1. Most of the cells were polygonal and triangular, the cells were positive for ALP with blue cytoplasm after staining at day14and the Alizarin red staining showed the formation of red mineralized nodules in the special mineralization induction medium at day28.2. Compared with the control group, Wnt2gene in G1r19(1-34) group was down regulated by2.045times, Wnt5b gene in G1R19(1-34) was upregulated by2.002times, and Wnt7b gene in G1R19(1-28) was downregulated by2.438times. These genes all involved in pathway in cancer.3. The expression of OC mRNA and Wnt5b mRNA was significantly higher than that of vehicle group (P<0.05);the expression of Wnt2mRNA was significantly lower than that of vehicle group (P<0.05); the expression of β-catenin mRNA in PTH (1-34) group was significantly higher than that of vehicle group (P <0.05); the expression of Wnt7b mRNA in PTH (1-34) group and G1R19(1-34) group was higher than that of vehicle group and the G1R19(1-34) group was higher than PTH (1-34) group and G1R19(1-28) group (P<0.05)CONCLUSION1. Signal-selective PTH analogs can affect different genes, including the Wnt-related factors2. In the Wnt-related factors, PTH (1-34) and G1R19(1-34) affect mainly canonical Wnt signal factors, but the G1R19(1-28) chiefly acts on non-canonical Wnt pathway.3. Wnt-related factors involved in cancer development pathway, which may be potential factors for osteosarcoma in mice caused by PTH.