The Preliminary Study Of CSP?-plus Modified Endostar Inhibiting Metastasis Of Hepatocellular Carcinoma

ObjectiveIn vivo and in vitro experiments to determine the role of rES-CSP in inhibiting HCC metastasis,and to explore the possible mechanism of rES-CSP in inhibiting HCC development and metastasis.So as to provide experimental basis and drug development ideas for HCC metastasisMethods1.The expression of rES-CSP recombinant protein by E.coli BL21(DE3)prokaryotic expression system and the soluble expression conditions(induction temperature and time)were optimized2.Purification of rES-CSP using HisTrapTM HP affinity chromatography column.The purity and concentration were determined by RP-HPLC and BCA kit4.Recombinant plasmid carrying the luciferase gene and GFP gene was transfected into HCCLM3 cells by lentiviral transfection method.A monoclonal cell line was stabally expressing luciferase and GFP were screened out by puromycin.And the activity of luciferase was detected by ATP fluorescence detector,the expression of GFP was observed by fluorescence microscopy.The highly metastatic hepatocellular carcinoma cell line expressing Luc gene and GFP gene was obtained.5.Liver orthotopic injection of Luc and GFP-labeled HCCLM3 cells in logarithmic growth phase to establish a nude mouse model of orthotopic liver cancer.6.The experiment was divided into:saline control group,Endostar group and rES-CSP group.The tail vein was administered two days once for 30 days.The in vivo imaging was used to observe the growth and metastasis of orthotopic tumors in nude mice in real time.7.After 8 weeks of imaging,the nude mice of each group were euthanized,and the growth and metastasis of orthotopic tumors were physical observed.The tumor mass was dissected by liver orthotopic to measure tumor volume and weight,and the tumor inhibition rate and lung metastasis rate were calculated.At the same time,the heart,liver,spleen,lung,kidney,and tumor of nude mice were taken for tissue sectioning.8.HE staining was used to observe the effects of rES-CSP on tumor morphology and toxic side effects of various organs in nude mice.9.Quantitative real-time PCR was used to detect the effect of rES-CSP on the mRNA expression of angiogenesis and metastasis-related genes in HCCLM3 cells.10.Western blotting was used to detect the effect of rES-CSP on the expression of angiogenesis and metastasis-related proteins in HCCLM3 cells11.The effect of rES-CSP on the expression of angiogenesis and metastasis-associated proteins in tumor tissues and metastases were observed by immunohistochemistryResult1.Obtaining rES-CSP soluble recombinant protein with high purity and concentration2.In vitro experiments showed that rES-CSP inhibited the proliferation,migration,invasion and adhesion of HCCLM3 cells compared with the control group,CSP I-plus and Endostar3.The identification showed that the HCCLM3 cell lines labeled with Luc and GFP were successfully constructed4.The nude mouse orthotopic liver cancer model was successfully established.The nude mice in the experimental group of orthotopic liver cancer were continuously observed in vivo imaging system.The fluorescence signal gradually increased with time.Compared with normal saline and Endostar group,the rES-CSP group had a significant decrease in abdomen luminescence;the solid observation showed that the inhibition rates of rES-CSP and Endostar group were 42.4.6 ± 5.39%and 11.1±1.88%,respectively.In addition,HE staining showed that the rES-CSP can promote tumor necrosis and inhibit tumor angiogenesis5.Tissue bioluminescence showed that the bioluminescence signal of liver tumor and lung metastasis was significantly attenuated in the rES-CSP group compared with saline and Endostar group.Physical observation showed that the lung metastasis rates of the control group,Endostar group,and rES-CSP group were 71%,50%,and 42.8%,respectively.In addition,HE staining results of lung metastases suggest that recombinant protein can inhibit tumor cell infiltration and growth.6.HE staining confirmed that rES-CSP had no obvious side effects on important organs of nude mice.7.Compared with the control group,rES-CSP down-regulated the expression of angiogenic protein VEGF in HCCLM3 cells,and up-regulated the expression of VEGF after the treatment of heparin sodium,a competitive inhibitor of CSP I-plus.Furthermore,rES-CSP down-regulated the expression of integrin?1 and MMP2 proteins in HCCLM3 cells,while E-cadherin protein expression did not change significantly.The expression of MMP2 and integrin?1 was up-regulated after HSPG heparin sodium.8.Compared with the control group,rES-CSP decreased the positive expression of VEGF protein,MMP2 and integrin?1 protein,but the positive expression of E-cadherin protein did not change significantly.There were no significant differences in the expression of E-cadherin,integrin?1 and MMP2 in the lung metastases.Conclusion:1.In vitro experiments confirmed that rES-CSP can inhibit the metastasis of HCCLM3 high metastatic liver cancer cells;2.A double-fluorescent labeled nude mouse model of orthotopic liver cancer was established.In vivo experiments confirmed that rES-CSP can inhibit the growth and metastasis of orthotopic liver cancer;3.It was preliminarily determined that rES-CSP may down-regulate angiogenic protein VEGF,metastasis-related proteins integrin?1 and MMP2 to inhibit tumor growth and metastasis,and may also inhibit the metastasis of liver cancer by interfering with HSPG-mediated tumor migration on the surface of hepatoma cells.