Experiments Studies On Inhibitory Effects Of Chimeric Molecule VEGI～+ On Human Malignant Lymphoma In Vivo

Objective1.To prepare the novel chimeric vascular endothelial growth inhibitor (VEGI+) with molecular biology methods.2.To establish the ideal animal mode of human malignant lymphoma and explore the inhibitory effect of chimeric molecule VEGI+ on malignant lymphoma in vivo.Method1 . Chimeric molecule VEGI+ was constructed by grafting oligopeptide CTTHWGFTLC to extracellular region of VEGI (VEGI23– 174).Before ligation into pET30a(+) expression vector,PCR product of this recombinant gene was cloned into pGEM-T vector and verified by restriction enzyme digestion and DNA sequencing.After transformation and expressed in BL21 (modified E. coli strain), the chimeric protein was purified by metal affinity chromatography.Western blots and coomassie blue staining then were used to confirm the protein.2.6~8-week-old nude mice were injected with cyclophosphane into abdominal cavity,100mg/kg,1/day,in all 2.Then the mice's backs were inoculated with human malignant lymphoma cell CA-46 subcutaneously.100% tumors grew after 2 weeks, and the nude mice transplanted tumor mode was prepared.20 nude mice with approximately same size tumors were chosen and divided into 4 groups randomly.4 groups'tumors were injected respectively with chimeric molecule VEGI+,pure VEGI,pure oligopeptide and PBS,1/day,period of treatment is 7 days.3.The curative effect of anti-tumor:We observed the animation of the nude mice and used sliding caliper to measure the long diameter (A) and short diameter (B) which crossed every tumors'center on 0,7,14,21,28 days after the first injection.We calculate the tumor volumes by the formula:(V)=A×B2/2 and the tumor inhibition ratio by the formula:Rate=(Volume of control group-Volume of therapeutic group)/Volume of control group×100%.4.Many methods like HE staining,TUNEL,immunohistochemistry,MVD in tumors , real-time fluorescence quantitative PCR and half quantitative reverse transcriptase PCR were applying to observe the pathologic changes,inhibition of tumor neovascularization and the expression of VEGF,MMP-2,MMP-9.Results1.The chimeric molecule VEGI+ was confirmed by restriction enzyme digestion and DNA sequencing.A chimeric protein about 23KD shows up in western blots and coomassie blue staining.Further purification yield chimeric molecule VEGI+ with purity about 90%.2.We have established the nude mice animal mode of human malignant lymphoma,and the nude mice survived well.All the tumors of 4 groups grew bigger gradually,but the chimeric molecule VEGI+ group grew slower evidently,the volumes of these tumors became smaller than the PBS group's obviously from the seventh day.The difference indicated statistical significance(P<0.01).The tumors'growths of pure VEGI group and pure oligopeptide group were inhibited too,the difference indicated statistical significance (P<0.05)compare with PBS control group.The chimeric molecule VEGI+ group demonstrated more significant tumor growth inhibition than pure VEGI group,pure oligopeptide group.And the pure oligopeptide group is better than pure VEGI group,simultaneity PBS group as control group.3.In chimeric molecule VEGI+ group,routine HE staining showed a lot of hyperplastic necrotic connective tissue and fibrosis,and a lot of apoptosis was observed by TUNEL(Terminal deoxynucleotidyl Transferasemediated dUTP nick end labeling).Immune histochemistry VEGF/CD34 staining showed that there are few expressings of VEGF , and the microvessel density is 26.3±4.5 . Real-time fluorescence quantitative PCR showed that the VEGF mRNA relative expressing rations is 1.0021±0.023.Half quantitative reverse transcriptase PCR showed that MMP-2 and MMP-9 mRNA relative expressing rations is 0.09±0.003 and 0.04±0.002.Above-mentioned differences in chimeric molecule VEGI+ group all indicated statistical significance compare with the other three groups(P<0.05).Conclusions1.We constructed the recombinant plasmid pET30a-VEGI+ and successfully expressed it in E. coli.A satisfied high purity chimeric protein VEGI+ was gained through affinity chromatography.2.The chimeric molecule VEGI+ can inhibit the growth of malignant lymphoma evidently . As an effective medication targeting to the vessels of malignant lymphoma,it has a good developing prospect.