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[Experimental study on the effect of hypoxia on the expression of VEGF and TGF-beta1 of rat mandibular osteoblasts].

OBJECTIVE
To observe the effects of cobalt chloride (CoCl2)-simulated hypoxia on VEGF and TGF-beta1 expression and to provide theoretical basis for deciphering the molecular mechanism of clinical distraction osteogenesis.


METHODS
The mandibular osteoblasts were obtained from newborn Wistar rats within 24 hours and cultured and purified through modified enzymatic digestion. The morphological and histological changes of cells were evaluated by the HE staining, the histochemical staining for ALP, the collagen I immunohistochemistry staining and the calcified nodules staining, and the growth curves were drawn. The best cells of the 3rd-passage rats were treated with CoCl2, and then immunofluorescence was used to detect the expressions of VEGF and TGF-beta1 at 0, 3, 6, 9, 12 and 24 hours after culture.


RESULTS
The HE staining demonstrated that the cellular forms were diverse, triangular, polygonal, circular and scaly and so on. The prominence varied in length and extended outwards. The nucleus was clearly discernible. The cytoplasma was rich and pink, with the nucleus royal purple. Sometimes 2 cell nuclei were seen. At the crowded place, cellular form was not clear, the dividing line was indistinct, and just the great-circle nuclear cells could be seen. The ALP immunohistochemistry staining demonstrated that the cell butcher nature appeared black pellets, the cell nucleus outline was unclear, and at the cell compact district, massive masculine cells could be seen clearly. The collagen I immunohistochemistry staining demonstrated that masculine cells were seen evenly, cytoplasma appeared yellowish brown especially around the nucleus. However, yellowish brown pellets were not seen in negative cells. The osteoblast calcium tubercle staining demonstrated that the cells gathered in the opaque region with the shape of tubercle after 15 days of culture. After alizarin red staining, the reddish orange pigmentation appeared. At various time points, weak VEGF fluorescence was seen in the cells in the control group under the laser confocal microscope. As the hypoxia time prolonged, VEGF fluorescence of cells in the experimental group intensified, and reached the peak 9 hours after operation, and then dropped to the normal level. At various time points, TGF-beta1 fluorescence was found in both groups under the laser confocal microscope, and fluorescence intensity in the control group was slightly stronger than that in the VEGF control group. In the experimental group, TGF-beta1 expression had short-term increase 3 hours after hypoxia, and reduced gradually with the prolonging of hypoxia time.


CONCLUSION
The method of culturing osteoblast from Wistar rats mandibular is practicable. The cells can be used for further studies. Moderate hypoxia can affect bone synthesis and turnover in distraction osteogenesis and up-regulate the expressions of VEGF and TGF-beta1.