Investigation Of Inhibitory Effect On Human Choriocarcinoma By Magnetic Nanoparticles Of Fe₃O₄-dextran-anti-βHCG For Antisense Oligodeoxynucleotide Of Heparanase

Partâ… Heparanase expression correlates with metastatic capability in human choriocarcinomaObjective:In order to investigate the association between the expression of heparanase(Hpa)and the metastatic capability of choriocarcinoma,we studied the expression of Hpa in human choriocarcinoma cell lines JEG-3,JAR and normal chorion.Methods:(1)The different invasion and proliferation ability between JEG-3 and JAR cell lines was proved by Matrigel invasion assay and MTT assay in vitro;(2)Reverse transcriptase polymerase chain reaction (RT-PCR),western blot were carried out respectively to determine Hpa gene and protein expression;(3)The localization of this molecule was demonstrated by immunohistochemistry.RESULT:(1)We proved that the invasion ability of JEG-3 cell line was stronger than that of JAR cell line(P＜0.05),but the difference of proliferation ability between JAR and JEG-3 is not obvious(P＞0.05);(2) We found that the Hpa gene and protein in JEG-3 and JAR cell lines were significantly higher than those in normal chorion(P＜0.05),On the other hand,we detected that JEG-3 expressed much more Hpa than JAR(P＜0.05);(3)Both in JEG-3,JAR cells and in normal chorion tissues,we found that Hpa protein express in cytoplasm;(4)The data suggested that there were significantly positive correlations between Hpa expression and the invasiveness of choriocarcinoma cells.CONCLUSIONS:The current study demonstrated the expression of Hpa plays an important role in metastatic capability of human choriocarcinoma. Partâ…¡Preparation and characterization of magnetic nanoparticles of Fe₃O₄-dextran-anti-βHCGObjective:magnetic nanoparticles of Fe₃O₄-dextran-anti-βHCG was synthesized by chemical co-precipitation method.To evaluate feasibility of using Fe₃O₄-dextran-anti-βHCG nanoparticles as gene carrier in the gene transfection of cell.Methods:(1)Fe₃O₄-dextran-anti-βHCG nanoparticles was synthesized by chemical co-precipitation method.The configuration, diameter and iron content of this nanoparticles was detected by transmission electron microscope,zetasizer and atomic absorption spectrophotometry;(2)MTT was used to evaluate the cytotoxicity of Fe₃O₄-dextran-anti-βHCG nano- particles;(3)ELISA and indirect immunofluorescence method was used to evaluate the immunoreactivity; (4)The potency of adsorbing gene and resisting DNaseâ… digestion of Fe₃O₄-dextran-anti-βHCG nanoparticles was analyzed by agarose gel electrophoresis;(5)The effect of gene transefection of Fe₃O₄-dextran-anti-βHCG nanoparticles and the ability of absorbing Hpa AS-ODN gene magnetic nanoparticies of different cell lines was detected by Flow Cytometry(FCM);(6)The target distribution of Hpa AS-ODN gene magnetic nanoparticies in transplanted choriocarcinoma tumor in nude mice was detected by atomic absorption spectrophotometry.RESULT:(1)Transmission electron microscope showed that shape of nanoparticies is irregular.Under zetasizer,the mean diameter of the Fe₃O₄-dextran-anti-βHCG nanoparticles is 75.5nm.The iron content of this nanoparticles is 375ug/ml;(2)The concentration of Fe₃O₄-dextran-anti-βHCG nano- particles no more than 37.5ug/ml have no cytotoxicity obviously;(3)Aldehyde Fe₃O₄-dextran nanoparticles have the satisfactory potential to combine withβHCG antibody;(4) Agarose gel electrophoresis of binding experiment shows that after treated by NaIO₄,Fe₃O₄-dextran-anti-βHCG nanoparticles have the satisfactory potential to absorb gene,and protection experiment shows that this nanoparticles can protect DNA from DNaseI digestion effectively;(5)Aldehyde Fe₃O₄-dextran anti-βHCG nanoparticles can transfect reporter gene into cells and the transfection rate of this nanoparticles is much more than that of liposomes(P＜0.05);(6) Fe₃O₄-dextran-anti-βHCG nano-particles can concentrate in choriocarcinoma cells and in transplanted choriocarcinoma tumor.CONCLUSIONS:The results confirm that Fe₃O₄-dextran-anti-βHCG nanoparticles can be a kind of secure,effective and choriocarcinoma targeting gene vector.Partâ…¢The study of inhibitory effect on choriocarcinoma by Fe₃O₄-dextran-anti-βHCG nanoparticles carrying antisense oligodeoxynucleotide of heparanaseObjective:The study is to observe the influence of Fe₃O₄-dextran-anti-βHCG carrying Hpa AS-ODN on the invasion,proliferation ability and the expression of Hpa of JEG-3 cells,on the transplanted choriocarcinoma tumor growth.Methods:(1)The different invasion and proliferation ability between transfected JEG-3 cells and untransfected JEG-3 cells was proved by Matrigel invasion assay and MTT assay in vitro;(2)The effect of Hpa AS-ODN transefection on the expression of Hpa mRNA and protein was measured by RT-PCR and western blot;(3)The transplanted choriocarcinoma tumors were taken out to calculate the inhibitory effect on tumor growth of Hpa AS-ODN.RESULT:(1)We proved that after transfected by Fe₃O₄-dextran-anti-βHCG carrying Hpa AS-ODN,the invasion ability of JEG-3 cells was inhibited efficiently(P＜0.05),but the difference of proliferation ability between transfected JEG-3 cells and untransfected JEG-3 cells is not obvious(P＞0.05);(2)We found that after transfected by Fe₃O₄-dextran-anti-βHCG carrying Hpa AS-ODN,the expression of Hpa mRNA and protein in JEG-3 cells was inhibited efficiently(P＜0.05);(3)Hpa AS-ODN has inhibitory effect on the transplanted choriocarcinoma tumor growth(P＜0.05);(4)Fe₃O₄-dextran-anti-βHCG carrying Hpa AS-ODN don't make side effect on nude mouse.CONCLUSIONS:According to inhibit the expression of Hpa, Fe₃O₄-dextran-anti-βHCG carrying Hpa AS-ODN weaken the invasion ability of choriocarcinoma cells,and produce inhibitory effect on the transplanted choriocarcinoma tumors.On the other hand, Fe₃O₄-dextran-anti-βHCG carrying Hpa AS-ODN is a secure gene therapy method.