Experiments Studies On Inhibitory Effects Of Chimeric Molecule VEGI~+-CS Nanoparticles On Human Malignant Lymphoma

Objective1.To prepare the novel chimeric vascular endothelial growth inhibitor(VEGI⁺) with molecular biology methods and its nanoparticles with chitosan.2.To choose chotisan as the drug carrier and establish the ideal animal mode of human malignant lymphoma and explore the inhibitory effect of chimeric molecule VEGI⁺-CS nanoparticles on malignant lymphoma.To explore the inhibitory effects of chimeric molecule VEGI⁺-CS nanoparticles on human malignant lymphoma.Method1.Chimeric molecule VEGI⁺ was constructed by grafting oligopeptide CTTHWGFTLC to extracellular region of VEGI(VEGI_23-174).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.To take the chotisan as the drug carrier,we get the VEGI+-CS nanoparticles, oligopeptide-CS nanoparticles,VEGI-CS nanoparticles.VEGI+-CS nanoparticles, oligopeptide-CS nanoparticles,VEGI-CS nanoparticles were prepared with chitosan as the drug carrier by inonotropic gelation process.3.6～8-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.35 nude mice with approximately same size tumors were chosen and divided into 7 groups randomly.Mice in each group tumors were injected with chimeric molecule VEGI+, pure VEGI,pure oligopeptide,VEGI⁺-CS nanoparticles,oligopeptide-CS nanoparticles,VEGI-CS nanoparticles and PBS,respectively for 7 days(once per day).4.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%.5.Many methods like HE staining,immunohistochemistry,MVD in tumors were applied to observe the pathologic changes and inhibition of tumor neovascularization.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 7 groups grew bigger and bigger gradually,but the tumors in VEGI⁺-CS-NPs group and VEGI⁺ group grew much slower evidently,and the volumes of these tumors became smaller than those in the PBS group's obviously from the seventh day.The differences indicated were statisticaly significancet(P＜0.01 =.The tumors' growths of growth of tumors in pure VEGI group and pure oligopeptide group were inhibited too,the differences indicated were statisticaly significancet(P＜0.05)compared with PBS control group(P＜0.05).The VEGI⁺-CS-NPs group and 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.Immune histochemistry VEGF/CD34 staining showed that there are few expressings of VEGF,and the microvessel density is 18.3±4.5.Above-mentioned differences in VEGI⁺-CS-NPs group,VEGI⁺ group all indicated statisticaly significancet compared with the other three groups(P＜0.05).Conclusions1.We constructed the recombinant plasmid pET30a-VEGI⁺ and successfully expressed it in E.coli.A satisfactory high purity chimeric protein VEGI⁺ was gained through affinity chromatography.2.The stability of VEGI⁺-CS-NPs in vivo is confirmed,and its effect of inhabition is better than other groups good.3.The VEGI⁺-CS-NPs 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.