A Preliminary Study On Transdifferentiations Between Osteoblasts And Adipocytes Of Rabbit In Vitro

Objective:Investigated the method and ability of transdifferentiation between osteoblasts and adipocytes, thus to prepared for the further research of the regulation by gap junction communication.Methods:Took adipose tissue from groin of 3 months New Zealand white rabbit, isolated and cultured the adipocytes. Using inverted ceiling adherent culture method to dedifferentiate the adipocytes, and the 3rd generation of the dedifferentiated adipocytes was used to carry out osteogenic induction for 3 weeks, and set control group for comparison. Examined the indicators of osteogenic differentiation:two groups of cells were treated with collagen type I immunohistochemical staining after cultured for 3 weeks; ALP active Assay Kit and BCIP/NBT Alkaline Phosphatase Color Development Kit were used to detect the ALP activity and ALT dying of each group cells at the 7th,14th and 21th day. Stained two group cells with alizarin after cultured for 3 weeks. Took the skull bone from neonate rabbit that born within 7 days, got osteoblast cells and cultured. The 3rd generation osteoblast cells were used for adipogenic-induction. And set control group for comparison. Examined the indicator of adipogenic differentiation:Oil Red O staining were used after cultur for 3 weeks. Detected the expression of PPARy mRNA by RT-RCR.Results:1.After adherent ceiling culture, the extracted mature adipocytes transformed from single-room round shape cells into a long spindle-shaped cells which is just dedifferentiated adipocytes.2. Examination of the osteogenic differentiation:the experiment group expressed type I collagen, and there was significant difference compared with control group(P<0.05). After examined by alkaline phosphatase activity assay kit, the ALP activity of cells in experimental group are higher than that in control group (p<0.05); by alkaline phosphatase staining kit, the experimental group were brown-black fine particles after staining, which were most distributed at the cell membrane and its surrounding. There was no brown-black fine particles in control group. By staining the calcium nodules of two groups with alizarin after consecutive cultured for 3-weeks, many orange nodules was found in the experimental group while little in the control group.3. After staining with alizarin, we could confirm that the cells we cultured were osteoblasts. 4. Examination of adipogenic-differentiation.:indentifing by oil red o staining, large amount of stained particles were found in experimental group while control group was not stained. The expression of PPARy mRNA was tested by RT-PCR, the result of the experimental group was positive, while the control group was negative.Conclusion:The mature adipocytes can dedifferentiat by external ceiling culture; The dedifferentiated adipocytes can differentiate to osteoblasts induced by osteogenic. Osteoblasts may also differentiate to adipocyte induced by adipogenic. The two type cells can convert to each other at certain condition.