| ObjectiveAdipose tissue is not only energy storage organization, Adipocyte secrete several regulatory factors, which ralate with diabetes, high blood pressure, atherosclerosis and inflammation, promote the occurrence and development of the cardiovascular disease. Insulin-like growth factor-1(IGF-1) like insulin, has variety bioactivities, which affect the proliferation, growth and differentiation of cells. IGF-1can also regulate the biological behavior of adipocyte, as a necessary factor for the period of G1to S. Focal adhesion kinase(FAK) regulate the proliferation of sereval cells, as a focal proint for the several signal transductions along with its family member. Especially PYK2which is a family member of FAK, regulate the cytoactive. IGF-1promote cell move, FAK phosphorylation, and activate PYK2to regulate cell adhesion. Gax is homeobox gene, which can inhibit cell migration, proliferation and promote apoptosis. Gax can stop the cell period of G1, which is an antagonist of IGF-1. We don’t know whether gax can inhibit the effect on the adipocyte’channel protein. This article in order to elaborate.Method1. Cut the object gene from the original plasmid. Gax was recombined with adenovirus vector. The right Ad-Gax was amplified and purified.2. Cell culture3T3-L1preadipocytes were prepared with the third generation or the forth generation. After preadipocytes get too confluent two days later, the preadipocytes were prepared by the"cocktail method".11days later,lipid accumulation were in the adipocytes. More than90%preadipoytes induced to muture adipoytes, which was determined by Oil Red O staining.3. Expriment Group (1)Containing Gax adenovirus:Ad-Gax transfected into the adipocytes. The adipocytes were divided into the normal control group, the Ad-GFP group and the Ad-Gax group. The intervention time was48h.(2)IGF-1intervention and Gax adenovirus transfection:The adipocytes were divided into the normal control group, the Ad-GFP group, the Ad-Gax group, the Ad-Gax+IGF-1group, and the Ad-GFP+IGF-1group. The intervention time was48h.4. The Western Blot The expression of gax at protein level were measured by western blot in the normal control group, the Ad-GFP group, and the Ad-Gax group. The expression of FAK,and PYK2at protein level were measured by western blot in the normal control group, the Ad-GFP group, the Ad-Gax group, the Ad-Gax+IGF-1group, and the Ad-GFP+IGF-1group.5. Quantitative Realtime Polymerase Chain Reaction The expression of FAK, PYK2mRNA were examined by qRT-PCR in the normal control group, the Ad-GFP group, the Ad-Gax group, the Ad-Gax+IGF-1group, and the Ad-GFP+IGF-1group.Results1. The Gax gene was successful connection with the adenovirus vectors, which was examined by the agarose gel electrophoresis.2. Adipocytes identification The preadipocytes were induced to the mature adipocytes. About5days, the liqid appeared.About11days, liqid appeared in more than90%adipocytes, which was detemined by Oil Red O staining.3. The recombinant adenovirus vector transfection Ad-Gax transfected into adipocytes48h later, more than90%adipocytes occure green fluorescent in the fluorescence microscope.4. The expression of protein The protein expression of Gax was increased obviously in the Ad-Gax group than the Ad-GFP group and the normal control group(p<0.01), after Ad-Gax transfecion48h later. The Ad-GFP group and the normal control group showed no significance difference(p>0.05). The protein expression of FAK, PYK2were increased clearly in Ad-GFP+IGF-1group than the normal control group, the Ad-GFP group, the Ad-Gax group and the IGF-1+Ad-Gax group(p<0.01). The protein expression of FAK, PYK2were reduced in Ad-Gax group, IGF-1+Ad-Gax group than the normal control group, the Ad-GFP group and the Ad-GFP+IGF-1group(p<0.01). The Ad-GFP group and the normal control group showed no significance difference(p>0.05).5.The expression of mRNA The mRNA expression of FAK, PYK2were increased clearly in Ad-GFP+IGF-1group than the normal control group, the Ad-GFP group, the Ad-Gax group and the IGF-1+Ad-Gax group(p<0.01).The mRNA expression of FAK, PYK2were reduced in Ad-Gax group, IGF-1+Ad-Gax group than the normal control group, the Ad-GFP group and the Ad-GFP+IGF-1group(p<0.01). The Ad-GFP group and the normal control group showed no significance difference(p>0.05).Conclusion1. Rat Gax gene can effectively transfect into the adipocytes and express in the adipocytes.2. Gax can inhibit the effect of IGF-1on the adipocytes.3. IGF-1can active FAK, PYK2signal transduction pathway, promote the expression of mRNA and protein, but the Gax gene can inhibit these fuctions. |
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