Effects Of Enhancing Or Inhibitting GJICon The Transdifferentiation From Adipocytes To Osteoblasts In Rabbits

Objective:To observe effects of GJIC enhancer PGE2or GJIC blocker18a-GA on transdifferentiation from adipocytes to osteoblasts and to explore the action of GJIC in transdifferentiation between osteoblasts and adipocytes. Discuss the regulator mechanism of GJIC in transdifferentiation between adipocytes and osteoblastsMethods:①Took the adipose tissue from groin of3months New Zealand white rabbit, abstract the MA from it with used mechanical separation and Enzyme Digestion.②Used inverted ceiling adherent culture method(culture flask filled with culture medium, turnover the culture flask make bottle up),which to obtain DA and subculture it.③Took the3rd generation of the dedifferentiated adipocytes which were used to carry out osteogenic induction (Osteogenesis inducers:DMEM culture solution with10%FBS,10mmol/Lβsodium glycero-phosphate,0.05mmol/LvitaminC and100mmol/Lhexadecadrol),added PGE2for GJIC intensifier and which is the strengthened group.Likewise,added the18a-GA for GJIC reducer and which is the weakened group,, and set control group for comparison. Used MTT method to draw the growth curvature of cells, calculated the time of cells multiplication, and then observed whether PGE2and18a-GA influence the multiplication of cells or not.④Examined the indicators of osteogenic differentiation:Stained three group cells with alizarin red after cultured for2weeks; Three groups of cells were treated with collagen type I immunohistochemical staining after cultured for2weeks;⑤Detected of GJIC:The express of Cx43was detected with western blot method; The function of GJIC was detected with SLDT method in every group; The structure and length of GJIC was detected with TEM.Results:①Primary cells of mature adipocytes appear small round and circular shape, morphological rule and gather distribution.②After the inverted ceiling adherent cultured,the extracted mature adipocytes transformed from single-room round shape cells into a long spindle-shaped cells which is just become dedifferentiated adipocytes in anoxic environment.③After using MTT method to draw the growth curvature of cells and calculating the time of cells multiplication, with the single factor analysis variance P=0.982.The difference was not significant (P>0.05), With the conclusion is that PGE2and18a-GA have no appreciable impact on cells’grow and multiplication.④The examination of the osteogenic differentiation:By staining the calcium nodules of three groups with alizarin red after consecutive cultured for2-weeks, the purple-red nodules were found in all groups. What’s more, in the examination of type I immunohistochemical staining, the result of three group of x±s115.804±8.476of the Enhancement Group,125.479±9.230ofthe Control Group, and134.551±7.301of the Inhibition Group. When comparing themto the P value (0.046and0.047in Control Group), there was a significant difference in statistics science between each two groups(P<0.05).⑤After using transmission electron microscopy to observe GJIC and measure the length and brightness of it. The x±S ofthe three groupsare:enhancement group2.705±0.109of the Enhancement Group,1.954±0.144of the Control Group,1.264±0.202of the Inhibition Group. After compared with control group respectively (P=0), there is a significance difference in statistics science.SLDT method was used to detect the function of GJIC in each group shows that the number of plies of LY spread Respectively is9-10layers in Enhancement Group,7-8layers in Control Group and4-5layers in Inhibition Group.Used Western blot method detect the express of Connexin43(Cx43) protein, scanned the X-ray photographic film with Chemi Imager5500V2.0software,used Fluor Chen2.0software to do the quantitative analysis,and detected the integrated density value,the results of x±S of three groupsare4826.69±1909.12in the Enhancement Group,48519.19±1678.50in the Control Group, and44158.92±1356.94in the Inhibition Group. After compared with control group P value,0and0.001, there was a significant difference in statistics science between each two groups(P<0.05).Conclusion:Mature adipocytes can turn to DA by ceiling adherent culture method in vitro.DA can differentiate the into osteoblasts in osteogenic induction condition, PGE2can enhance GJIC and result in enhance the capability of differentiate to osteoblasts,18a-GA can inhibit GJIC and result in inhibit the capability of differentiating the osteoblasts.