Research Of The Inhibitory Effect On The Proliferation Of Perivascular Adipocyte By Gax

Objective:To establish a recombinant adenovirus vector for Gax gene using the adxsi system.Then observe its expression in mouse perivascular adipocyte and explore the effects of Gax on the proliferation of mouse perivascular adipocyte.Methods:1. Construct Gax adenovirus expression vector:the mouse Gax gene was amplified by PCR method. Obtain pAd-Gax by inserting the gene of interest into the shuttle vector. Then the recombinant adenovirus vector was transfected into293cells. Recombinant adenovirus Ad-Gax which coding for Gax gene was successfully constructed. Purify and concentrate Ad-Gax.2. Preadipocyte (3T3-L1) were cultured anchorage-dependent, and subcultured when they reach to80%confluence in petri dishes.2-6generation cells can be used in this experiment.3. While the2-6generation3T3-L1cells are available, culture the cells anchorage-dependent. Induce cell-differentiation using inducer IBMX, insulin and dexamethasone when3T3-L1cells reach to100%confluence of culture vessels.8days later, preadipocyte differentiate into adipocyte, which can be identified by oil-red O staining.4. The adipocyte were randomly divided into six groups:Ad-GFP group, Ad-GFP+IGF-1group, Ad-Gax+IGF-1group, Ad-Gax group, IGF-1group and normal control group(PBS group). The final concentration of IGF-1in culture medium is80ng/ml. 5. Transfection and GFP-detection:Virus-suspensions (the MOI of Ad-Gax and Ad-GFP are both100) which is prepared using Opti-MEM is used to culture adipocyte. After5-6hours incubation at37℃, add common induce-medium and cells were continue to be cultured overnight and change into common induce-medium for24h,48h,72h respectively. Then observe the transfection-efficiency which can be reflexed by GFP.6. The expression of ERK2protein at protein level after effect of Ad-Gax and IGF-1was examined by Western-blot.7. RT-PCR:Observe the RNA expression of ERK2protein after intervention of Ad-Gax and IGF-1.Analyses were performed using the statistical software package SPSS18.0.Results:1. PCR and sequencing indicated that the target fragment of Gax gene had been transferred into the denovirus vector correctly. The recombinant Ad-Gax was successfully constructed and it was identified by western blot anlalysis. The titer of Ad-Gax was1.6×1011pfu/ml.2. Induce preadipocytes cell-differentiation when contact-inhibition. After three days of induction, lipid droplets appeared. The differentiation rate was more than80%after eight-day induction. Oil-Red O staining confirmed the differentiation.3. The efficiency of Ad-Gax infecting mouse adipocytes was highest at72h after transfection, and it was about80%-90%. Almost all of the mouse adipocytes died after7days of transfection.4. Western-blot results:Compared with the Opti-MEM group and the Ad-GFP group,the proliferatin of the adipocytes transfected with Ad-Gax showed significant decrease at72h after transfection (P<0.05). The Ad-GFP group and the Opti-MEM group showed no significant differences (P>0.05); Compared with the Ad-Gax group and the Ad-Gax+IGF-1group, the proliferatin of the adipocytes transfected with Ad-Gax+IGF-1showed significant increase (p<0.05).5. RT-PCR results:Compared with the Opti-MEM group and the Ad-GFP group,the channel protein mRNA level of the adipocytes transfected with Ad-Gax showed significant decrease at72h after transfection (P<0.05). The Ad-GFP group and the Opti-MEM group showed no significant differences(P>0.05); Compared with the Ad-Gax group and the Ad-Gax+IGF-1group,the channel protein mRNA level of the adipocytes transfected with Ad-Gax+IGF-1showed significant increase (p<0.05).Conclusion:1. We have constructed the recombinant Ad-Gax successfully using Adxsi system and packaged it into maturated denovirus. Compared to the traditional system, it has advantage of efficiency and convenience, which lays an experimental foundation for the future research on the physiological function and genetic therapy of Gax.2. It’s the adipocytes, which were from preadipocytes by certain induction technology, that be used in the experiment. Compared to using primary fat cell, it’s more applicable and convenient. The improved induction technology led to much higher differentiation-efficiency. This lays an experimental foundation for the future research of adipocytes.3. Ad-Gax could transfect perivascular adipocyte (PVAC) effectively. Compared to the liposome transfect method, it has higher efficiency.4. The over-expression of Gax could suppress the proliferation of perivascular adipocyte.