Biological Mechanism Of Action Of Heparanase On Invasion And Metastasis Of Human Epithelial Ovarian

The morbility of epithelial ovarian is the third in malignant gynecology tumors while the mortality is the first.Ovarian conceals cavum pelvis deeply.On the pathogenetic early stage, deficiency of characteristic symptom and physical symptom make the discovery and diagnosis difficult.About 60-70%of ovarian cancers is advanced stage when they are diagnosed.In recent 30 years,chemotherapeutics modus were discovered,and operandi has been modified.but the 5 years survival rate is just 20-30%.Finding new diagnostic method and therapeutic tool is the focal point in research of ovarian cancer.Heparanase is associative with infiltration of tumor intimately.Heparanase plays an important role in ovarian cancer infiltration too.We discussed the mechanism and effect of heparanase on invasion and metastasis of human epithelial ovarian in vitro.And then we constructed lentivirus expressing vector and RNAi vector of HPSE gene.That can promote the gene targeted therapy. Amplification and eukaryotic vector construction of heparanase gene fromhuman ovarian cancer tissueObjective The goal of this study was to amplify the full length of human heparanase gene and construct a eukaryotic vector so as to facilitate the further study.Methods We extracted total RNA from mucus ovarian cancer tissue and then performed reverse transcription polymerase chain reaction to amplify the full length of heparanase gene.The amplicon was then cloned into the vector of pcDNA3.1.The result was confirned by enzyme digestion and sequencing.Results We successfully amplified the full length of human heparanase gene and cloned it into the vector of pcDNA3.1.The homology was 99.9%confirmed by sequencing.Conclusion We successfully amplified the full length of heparanase and cloned it into eukaryotic vector.Mammalian:heparanase ovarian cancer eukaryotic vectorAn in vitro study on heparanase mediated invasive ability of epithelial cancerObjective:To study heparanase on ovarian function in cancer cells,and analysis its affection on ovarian cancer invasive and metastasis,and pilot its role in early diagnosis, treatment targeting treatment.Method:We successfully constructed HPSE gene expression vector,and transfered it into ovarian cancer cell line A2780,After G418 selection then screening by RT-PCR and western-blot to confirm a stable transfer of the cell lines HPSE/A2780.Then proceed to test the growth of cells,cell cycle,the capacity of adhesion, cell invasion and metastasis experiment.Results:after transfer and screening,we succefully stabilize the protein expression HPSE in ovarian cancer cell line.FCM test showed that A2780-HPSE is in a proliferation of state(S + G2 + M)for 46.5%,G1 for 53.5%, corresponding to the control group,54.5%and 45.6%,respectively.HPSE group and the control group the growth curve almost coincidence.The difference of clone forming unit of the two groups was not statistically significant.A2780 cell adhesion before and after the transfer rate was 0.5183±0.0796 and 0.7283±0.08931,There was significant(p = 0.002). Transwell small room found that gene transfer HPSE after the invasion of A2780 cells markedly improved,compared with the control group were significant(p = 0.003).And its migration were not affected(p = 0.618).Conclusion:cell functions test showed that HPSE can enhance the invasive ability of A2780 ovarian cancer cell,reducing its adhesion ability of the proliferation and migration were not affected.Mammalian:heparanase transfer ovarian cancer cell functions Objective:We constructed the expression vector of shRNA and transfer it into ovarian cancer cell lines,then test its interfere rate on HPSE gene function.We also screened some stable transfer cells and did series gene function test.Method:We designed several shRNA vector to inhibit various HPSE mRNA domains and lipofect them into SKOV3,in which HPSE is highly expression.We use real-time PCR to test the interfere rate and we test the antibiotics resistance of the highest interfere group.And we got the stable cell line and did western-blot to test the inhibition of HPSE protein.Results:fluorescence quantitative PCR to test different shRNA interference vector interference effects show that,pGPU6/GFP / Neo-HPSEo1222 goup reative copy number is 0.936±0.417,having statistically significance compared with the other three groups,inhibition rate is 48.63%.The group was stable interfere and western-blot test the expression of HPSE decreased significantly.The cell growth curve showed that cell doubling time extended.Interference group and control group colony-forming rate was 0.0433±0.0451 and 0.0397±0.00687,p = 0.059 and was not statistical significant.In the Invasion experiment,the interference groups adherence value was 0.5697±0.106,the control group was 0.8097±0.333,interference and control group was not significant(p = 0.233).Cell adhesion ability test showed that interference group was 0.7533±0.07685,the control group was 0.5833±0.11994,having statistically significant(p = 0.015). Invasion of interference group was 0.69±0.085,the control group was 1.091±0.277,two groups showed statistically significant(p = 0.015).Conclusion:we successfully constructed shRNA interference HPSE gene vector,effectively curbed HPSE gene activity,reduced the invasion and metastasis ability of ovarian cancer cell.Mammalian:heparanase RNAi shRNA interference vector cell functions Objective:Using the second and thkd generation lentivkal vector to construct HPSE expression lentivirus vector and shRNA interference lentivirus vector,exploring a more efficient and reliable method than traditional method of gene transfer,laying a good foundation for further research of targeted therapy.Method:First of all we amplified HPSE full-length sequence,and cloned it into lentiviral vector pWPI and them sequencing,and used BLAST to search the similarity.We transferred reconstruction lentivirus plasmid into 293 T cells,after 48 hours extracted mRNA,RT-PCR test HPSE gene expression.Second,we selected the best interference siRNA,and design shRNA,annealing to a double-stranded and cloned into lentiviral vector pSico then sent to sequencing.Results:BLAST searched the reconstruction of lentivirus vector HPSE-pWPI sequencing results,we found that homology of the vector sequence and HPSE gene is 99%.293 T cell line transfected by HPSE gene can expression HPSE protein.The recombinant of the slow-interference vector pSico / HPSE sequence and the design of shRNA sequence is exactly the same.Conclusion:we successfully constructed the HPSE lentivirus system and the lentivirus interference system laying the foundation for further study of HPSE targeted therapy.Mammalian:heparanase lentiviralvector transfer siRNA...