| Several recent findings have demonstrated that vitamin D plays a role as a factor for cell differentiation, function and survival. In bone, vitamin D stimulates bone turnover while protecting osteoblasts of dying by apoptosis whereas in muscle vitamin D maintains the function of type II fibers preserving muscle strength and preventing falls. Osteoblasts, the main functional cells for bone formation, are responsible for the synthesis, externalization, and mineralization of bone matrix. Isolating and culturing osteoblasts in vitro provides conditions for toxicology, pharmacology, physiology and gene engineering. High purity and vitality osteoblasts are the premise for study. To study the effects of vitamin D3 injection on osteoblasts, osteoblasts were isolated from novo-rat cranial bones and cultured in vitro. In addition, influences of vitamin D3 injection on Silkie laying rate were studied through intramuscular injection. These results provided rationale for clinical application.1 Isolation and identification of rat osteoblastsTo obtain osteoblasts, cranial bones were harvested from novo-rat, digested by trypsin and collagenase. Then the cells were brachytely centrifuged for purify and seeded to culture flask. The culture medium replaced after 24 h to remove un-adherence cells. Shape and growth of OB were observed under inverted microscope. They were identified by ALP stained and mineralizated node stained. The activity of osteoblast was detected by microtitration (MTT) during a culture cycle. It was concluded that the modified twice-enzyme isolation method was an ideal technique to obtain and culture osteoblasts with typical characteristics and high purity for the experiment.2 Effects of vitamin D3 on the osteoblasts cultured in vitroIn order to observe the proliferation and functions of vitamin D3 on the osteoblasts in vitro, different concentrations of vitaminD3 injection (0,10-10,10-9,10-8 mol/L) were added into culture solution. Then cell proliferation was observed by microtitration (MTT), ALP activity was detected through PNPP method, and the number of mineralized node were counted after stained by von Kossa. The results indicated that, compared to control group, 10-9 and 10-8 mol/L vitamin D3 injection promoted proliferation of osteoblasts from the 1st day to the 3rd day (P<0.05 or P<0.0). A few days later, each group of vitamin D3 injection turned to inhibit the proliferation of osteobalst, but had no significant difference (P>0.05). Compared to control group, each concentration of vitamin D3 injection promoted ALP activity significantly or very significantly (P<0.05 or P<0.01). While the 10-10, 10-9 mol/L vitamin D3 injection markedly promoted formation of mineralized node (P<0.05) (in size and number). It was concluded that vitamin D3 injection could enhance bone formation of osteoblast, 10- 9 mol/L vitamin D3 injection was the best one.3 The influences of vitamin D3 injection in egg laying performanceTo study the influence of vitamin D3 in laying rate, blood calcium and phosphorus, Silkie breeding birds were injected with different dosage of vitamin D3 (1000 IU, 2000 IU, 4000 IU/hen). The results showed that the egg laying of control group (0 IU) kept stabilizing, and the 1000 IU group presented a tendency of increasing, and the 4000 IU group showed a tendency of degression. Compared to control group,the blood calcium content of groupâ…¡(1000 IU) steped up significantly (P<0.05),the blood phosphorus content in each group all increased significantly (P<0.05). Compared to the control group,the mean Ca:P ratio in groupâ…£(4000 IU) degraded significantly (P<0.05). These results suggested that intramuscular injection with 1000 IU vitamin D3 could elevate the blood calcium, blood phosphorus and laying rate of Silkie.
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