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Parathyroid hormone (PTH) synthesis and secretion by the parathyroid chief cells, regulating calcium and phosphorus metabolism and bone turnover peptide hormones. The body's blood circulation, the the three PTH molecule: PTH1-84 PTH37-84 and a small amount of PTH7-84, the latter two are called PTH carboxyl terminal peptide (C-PTH). The study found that PTH1-34 with identical PTH1-84 receptor binding capacity and osteogenic role, the U.S. FDA for the treatment of post-menopausal women was approved in 2001, parathyroid hormone (1-34) peptide (rhPTH1-34) After osteoporosis. It was found that when the rats were given PTH1-34 significantly increased incidence of osteosarcoma (highest tumorigenic rate of 52%). Recently found that human memory can produce biological effects of proapoptotic C-PTH and its specific receptor (PTH receptor) binding in the C-PTH receptor (CPTHR). Therefore, we infer PTH1-34 into bone - that is, anti-osteoblast apoptosis may induce tumor formation, and pro-apoptotic role of C-PTH inhibit the tumorigenic effect of PTH1-34. The inference, if established, will produce a far-reaching impact of PTH in the regulation of bone metabolism, the role of research. The aim of the present study intends to explore from both cell and animal experiments, using real-time PCR, western blot experimental techniques and methods PTH1-84, PTH37-84, and PTH1 3 4 on cell cycle . Period to clarify the different roles of the different PTH molecule osteoblast apoptosis, in particular, the human body contains the C-PTH molecule that PTH37-84 on the cell cycle and apoptosis of osteoblasts impact, thus further prove that the C-PTH The pro-apoptotic role, may be related to the inhibition of tumorigenesis. Method 1. Primary cultured osteoblasts: 2-day-old rats, the enzyme alternately digestion method osteoblasts were separated. ALP staining, Von Kossa staining were identified. 2 for drug intervention: Add 10 9 mol / L, PTH1-34, PTH37-84, PTH1-84, the control group was added an equal volume of PBS, and cultured for 24h, cells were harvested, total RNA and protein were extracted 3. real-time PCR technology ,2-ΔΔCT method to detect apoptotic genes osteosarcoma: of bax, bcl-2, of caspase3, c-fos of cyclin Dl mRNA levels, and detection of the housekeeping gene GAPDH as internal reference. SPSS 14.0 statistical software processing test data, multiple group comparison using one-way ANOVA, pairwise comparisons using the Student-Newman-Keuls test. 4. osteosarcoma tumor detected at the protein level using western blot technique, apoptotic protein (detected object ibid.), and detection butler gene GAPDH as reference to the exposure X-ray photo UVP gel imaging analysis system scan to calculate the ratio of the optical density of each gene protein bands with the internal reference GAPDH protein bars, projected trends expressed by various apoptotic factors. 5 to build an animal model: combined with resection of the thyroid and parathyroid glands of rats were randomly divided into four groups, were given 40μg/Kg PTH1-34-84 PTH37 PTH1-84 and saline, administered for 2 months take bone tissue were extracted total RNA and protein. 6. using real-time PCR, western blot detection of osteosarcoma tumor-related apoptosis gene expression at the mRNA and protein level (the same cells in experimental part). cells in the experimental part: (1) identification of primary osteoblasts: primary osteoblast cells were passaged observed under a microscope, cells adherent growth, morphology to irregular-shaped, long spindle, triangle mainly by the ALP staining, indicating that cells high purity and Von Kossa staining, mineralized nodule the endoscopic see black, this experiment was isolated primary osteoblasts mineralization. (2) PTH1-34 on primary osteoblasts: Experimental results show that given 10-9 mol / L PTH1-34 primary osteoblast cells, inhibition of apoptosis genes (such as Bcl-2, of caspase3, c-fos of cyclin Dl) compared with the control group, the expression increased significantly; promotion of apoptosis (e.g., bax) gene expression was significantly reduced as compared with the control group, the results of the mRNA level and protein level. for primary osteoblasts effect (3) PTH37-84: Experimental results shown, given 10 9 mol / L, PTH37-84 primary osteoblasts, and promote cell apoptosis (e.g., bax) gene expression was significantly increased as compared with the control group,; genes that inhibit apoptosis (e.g. Bcl -2 of caspase3, c-fos, cyclin Dl) and the control group compared to the expression level was significantly reduced. mRNA levels and protein levels were consistent. Animal experiments: (1) successfully established animal models. (2) PTH1-34 on rat osteoblast role: to give 40μg/Kg PTH1-34 rat detection results of the cell experiment, and the results of the mRNA and protein levels. Role: to give (3) PTH37-84 on rat osteoblast the 40μg/Kg PTH37-84 rat detection results of the cell experiment is consistent, and the results of the mRNA and protein levels. Conclusions We successfully cultured primary rat osteoblasts. Cell experiments confirmed 10-9 mol / L concentration PTH1-34 may inhibit rat primary osteoblast apoptosis. 1O-9 mol / L concentration PTH37-84 may have a role in promoting osteoblast apoptosis in rat primary. We successfully constructed the removal of the thyroid and parathyroid animal model. Animal experiments confirmed 40μg/Kg the PTH1 3 4 may have a role in inhibition of rat osteoblast apoptosis. 40μg/Kg PTH37-84 may have a role to promote apoptosis in rat osteoblasts. Cell and animal experiments are preliminary confirmed PTH1-34 may have a role in inhibition of apoptosis, and PTH37-84 has a role in promoting apoptosis.
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