Construction Of Adenovirus Vector Mediated Hyperplasia Suppressorgene And Experimental Studies On Inhibition Of Glioma On Rat By It

Objective To construct a a recombinant adenovirs encoding rat hyperplasia suppressor gene (rHSG) and study the inhibitory proliferation effect of it on the angiogenesis and growth of glioma in rat. Methods (1) the rHSG gene was extracted by enzymaticcuting the used pGM-T-rHSGwas construsted and identified in previous experiment and subcloned in the Shuttle-CMV-EGFP plasmid.Then the rHSG gene was transferred from pShuttle-rHSG to pAdxsi viral DNA by double enzymatcuting obtain the recombinant plasmid pAdxsi-GFP-rHSG,the recombinant pAdxsi-GFP-rHSG was selected by kansmycin resistanc and restriction endonuclease;(2) After linearized by PacI,the recombinant adenovirus DNA pAdxsi-GFP-rHSG was transfected into 293 cells for packaging and amplification ,Adxsi-GFP-rHSG was further identified. (3) A glioma model was established by injection of C6 cells into the right caudate nucleus of Sprague-Dawley rats by stereotaxic guidance. NS(A),Adv–rHSG–GFP(B) and Adv–GFP(C) were injected into the tumors on d 4 and d 11.we measured tumor size. (4) The PCNA, rHSG and MVD were detected by using immunohistochemical method. The expression of rHSG mRNA were detected by semi-quantity RT-PCR method。The expression of rHSG proteins were detected by Western-blot analyses。Results (1) We have successfully constructed recombinant adenovirus encoding rat hyperplasia suppressor gene (rHSG) Adxsi-GFP- rHSG,the expression of it on rat C6 brain glioma cells was more effective. (2) Group B could shrink its volume significantly, there were statistically significant difference than group A and C (p<0.01). (3) The microvessel density(MVD)was significantly lower in groups B than group A and groups C, there were statistically significant difference than group A and C (p<0.01). (4) At 9 ,15 ,21 days of experiments, for group A the expression of rHSG mRNA were separately 12.63±1.83,10.42±2.25,6.95±1.18. and for group B were 20.15±1.80,50.08±1.48,35.37±2.52. and for group C were 12.65±2.04,10.10±2.04,7.95±0.97. Comparing with group A and group C, the expression of rHSG mRNA in group B were significantly upregulated, and peaked at 15 days,there were statistically significant difference than group A and C (p<0.01). (5) At 9 ,15 ,21 days of experiments, for group A the expression of rHSG proteins were separately 73.77±2.70,56.72±5.12,43.52±1.81. and for group B were 73.06±2.99,117.82±5.37,94.02±1.45. and for group C were 75.09±2.83,53.15±2.60,42.06±3.02. Comparing with group A and group C, the expression of rHSG protein in group B were significantly upregulated, and peaked at 15 days,there were statistically significant difference than group A and C (p<0.01). Conclusions We have successfully constructed recombinant adenovirus encoding rat hyperplasia suppressor gene (rHSG) Adxsi-GFP- rHSG,the expression of it in rat C6 brain glioma was more effective. In vivo studies,injection of it into tumor significantly inhibited the growth of C6 brain glioma,and the MVD were significantly lower too in the experiment groups than those in the control group,the anti-tumor effect of rHSG may arise from both anti-angiogenesis and anti-proliferation effects.