| Objectives:1.To detect the positive expression rate of Golgi membrane protein 73(GP73)on the surface of hepatoma cells and other non-hepatocellular carcinoma cells.The specificity of GP73 expression on the surface of hepatocarcinoma cells was determined.2.Prepare common cationic liposomes.3.Cationic Liposome was conjugated with GP73 antibody to construct a novel Anti GP73-specific targeted vector for hepatoma cells.4.The transfection efficiency of GP73 antibody modified cationic liposomes containing different doses of GP73 antibody on the Hep G2 cells was investigated.Flow cytometry was used to screen the group with the highest transfection rate and the optimal load was determined.5.To compare the transfection efficiency of Anti GP73-modified cationic liposomes and common liposomes on human hepatocellular carcinoma cell lines Hep G2,Bel-7402,SMMC-7721,human normal hepatocytes HL-7702,human non-hepatocellular carcinoma SW480 and normal human embryonic kidney cells HEK293 T.6.To detect the targeted killing effects of Anti GP73-lipoplexes/HSVtk/GCV system on hepatocellular carcinoma cells.7.To investigate the killing effect of Anti GP73-modified liposome-mediated HSVtk suicide gene combined with GCV on tumor-bearing nude mice.Methods:1.Hep G2,SMMC-7721,Bel-7402,HL-7702,Hela,SW480 and HEK293 T wereincubated with anti-GOLPH2 and secondary antibody.The positive expression rate of GP73 was detected by flow cytometry.2.Preparation of common cationic liposomes using modified ethanol injection method and extrusion method(pre-laboratory preparation method).3.The GP73-modified cationic liposomes were prepared by coupling the GP73 antibody with the liposomes.4.The common cationic liposomes and liposomes modified by GP73 antibody were incubated with p Genesil-1 to determine the optimal mixing ratio of GP73 and liposome.5.Different doses of GP73 antibody was conjugated with the common cationic liposomes,which then were used to transfect Hep G2,Bel-7402 and SMMC-7721 cells.Transfection efficiency was detected by flow cytometry to determine the optimal load on GP73.6.Hep G2,Bel-7402,SMMC-7721,HL-7702,Hela,SW480 and HEK293 T cells were transfected with cationic liposomes modified by GP73 antibody to deliver p Genesil-1plasmid,and the expression of EGFP report gene was detected by flow cytometry.7.Hep G2,Bel-7402 and HL-7702 cells were transfected with cationic liposomes modified by GP73 antibody to deliver p Survivin-TK-PBI-CMV2 plasmids.After 48 hours,total RNA and protein were extracted from the cells.The m RNA and protein levels of HSVtk gene were detected by RT-PCR and Western blot.8.Hep G2,Bel-7402 and HL-7702 cells were transfected with cationic liposomes modified by GP73 antibody to deliver p Survivin-TK-PBI-CMV2 plasmids.Then adding150 ?g/m L precursor drug GCV for 24 h,and the apoptosis was detected by flow cytometry.9.Graded doses of GCV were treated with transfected hepatoma cells for 48 h.MTT assay was used to detect the effect of HSVtk suicide gene system on the survival rate of liver(cancer)cells.10.Hep G2 cells were inoculated with nude mice.The tumor volume was calculated using the formula V=ab2/2,(a represents the longest diameter of the tumor,and brepresents the shortest diameter of the tumor).11.HE staining was used to detect the necrosis of tumor tissues.12.TUNEL apoptotic kit was used to detect the apoptosis of three groups after intervention.13.The growth status of nude mice after treatment was observed and the survival status of nude mice was recorded for 40 days.The data was analyzed by Graphpad Prism v 6.0 software,and the growth curve was figured.The survival time of the three groups was compared.Results:1.After screening the positive expression rate of GP73 on the cell surface,the positive rate of GP73 on Hep G2,Bel-7402 and SMMC-7721 cells was higher than HL-7702,Hela,SW480,HEK293 T cells,and the difference was statistically significant(P<0.05).It indicated that GP73 is specific on the surface of hepatocarcinoma cells.2.A common cationic liposome was prepared.3.A GP73 antibody-modified targeted vector for hepatocarcinoma cells was prepared(Anti GP73-lipoplexes).4.The transfection efficiency of GP73 antibody-modified cationic liposomes on human hepatoma cell lines Hep G2,Bel-7402 and SMMC-7721 was significantly higher than that normal liver cells and non-hepatocellular carcinoma cells(P<0.05).5.The result showed that HSVtk gene was expressed in Hep G2 and Bel-7402 hepatoma cells,but not in normal liver cells HL-7702.6.The MTT results showed cell proliferation rate was lower in the transfection group than in the non-transfection and normal liver cells groups(P<0.05),and the viability of transfected cells decreased with the increase of GCV dose.7.The apoptosis rate of Hep G2 and Bel-7402 cells were significantly higher than that of normal liver cells(P<0.01).8.Liver tumor model in nude mice was successfully established.9.Immunohistochemistry and TUNEL detection of tumor tissue apoptosis showed that the apoptosis rate of tumor tissue cells was obvious in the Anti GP73-lipoplexes+HSVtk + GCV treatment group,while the lipoplexes+HSVtk +GCV group only had a small amount of apoptosis and no apoptosis was observed in the lipoplexes+Vector+GCV group.10.Survival curve analysis showed that the long-term survival time in Anti GP73-lipoplexes+HSVtk +GCV group was longer than that in control group(P<0.05).Conclusions:1.The positive expression rate of Golgi membrane protein GP73 on Hep G2,Bel-7402 and SMMC-7721 cells was higher than that of normal liver cells and non-hepatocarcinoma cells.2.GP73 antibody modified cationic liposomes(Anti GP73-lipoplexes)deliver transport system was successfully constructed.3.Anti GP73-lipoplexes has a high transfection efficiency for hepatocarcinoma cells.4.The Anti GP73-lipoplexes mediated plasmid p Survivin-TK-PBI-CMV2 successfully expressed in Hep G2 and Bel-7402 cells with high expression.5.At the cellular level: GP73-lipoplexes mediated SUR promoter-regulated HSVtk/GCV suicide gene system has a cytotoxic effect on Hep G2 and Bel-7402 cells and has no cytotoxic effect on normal hepatocytes HL-7702.6.Hep G2 hepatocarcinoma cell xenografts were inoculated successfully in nude mice.7.At the animal level: Anti GP73-lipoplexes mediated SUR promoter-regulated HSVtk /GCV suicide gene system inhibited the growth and proliferation of Hep G2 hepatocellular carcinoma xenografts in nude mice,and can also prolong the survival time of the nude mice in the treatment group. |
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