The Study On The Relation Between Caveolin-1 And Biological Behaviour Of The Cervical Cabcer Cell Lines After CO₂ Pneumoperitoneum

THE STUDY ON THE RELATION BETWEEN CAVEOLIN-1 AND BIOLOGICAL BEHAVIOUR OF THE CERVICAL CABCER CELL LINES AFTER CO2 PNEUMOPERITONEUMObjective : To study the relation between caveolin-1 and biological behaviour of the cervical cancer cell lines after CO2 pneumoperitoneum .Methods: Obtain cervical cancer Hela cell lines which overexpression and lower expression caveolin-1.Results: In contrast to the control group, overexpression caveolin-1 can restriain the cervical cancer Hela cell line ability of the growth , proliferation , invasion and metastasis,highexpression caveolin-1 can accelarate the cervical cancer Hela cell line ability of the growth , proliferation ,invasion and metastasis.Conclusion: Caveolin-1 is gene of tumour restraining,the expression content is reduce in cervical cancer cell line Hela after CO2 pneumoperitoneum, maybe accelarate its ability of the growth, proliferation ,invasion and metastasis. Construction and expression of PEGFP-N1 eukaryotic expression vector containing caveolin-1Objective: To clone the cDNA sequence of caveolin-1(CAV1) gene which contains the opening reading frame and construct the eukaryotic expression vector of PEGFP-N1/CAV1.Methods:The primers were designed and synthesized with enzyotomic sites and protective basyls and the caveolin-1 gene was obtained by RT-PCR,vector PEGFP-N1 and caveolin-1 gene were double digested by internal restriction enzymes, the sectioned fragments of PEGFP-N1 and caveolin-1 were retrieved after electrophoresis, the eukaryotic expression vector of PEGFP-N1/CAV1 was then constructed by a coupled reaction.Results:A fragment weighed about 555bp appeared after the recombinant plasmid PEGFP-N1/CAV1 was electrphoresized by PCR ,the recombinant plasmid was proved to release a piece of fragment weighed 555bp and a vector fragment weighed 4.8kbp from the electrophoresis results after double digested by internal restriction enzyme,the sequencing results was 100% accurate by contrast of BLAST .Conclusion Successfully constructed the eukaryotic expression vector PEGFP-N1/CAV1 which carried with both green fluorescence protein and G418 screening site. Over expression of caveolin-1 in cervical cancer Hela cell line and study its biological behavior in vitroObjective: To investigate overexpression of caveolin-1 in cervical cancer Hela cell line and study its influence on growth , proliferation ,infitration and metastasis of cervical cancer cell Hela .Methods :The eukaryotic expression vector PEGFP-N1/CAV1 was transfected into Hela cells by lipofectamine , obtaining colones which stably over expression caveolin-1 by G418. identified by real-time PCR. Test the growth ability by the FCM,growth curve,cell colony formation test ;and test the infitration and metastasis ability by the invision assay, migration assays, adhersion assay .Results :The expression vector of caveolin-1 was successfully constructed and the clony which stably overexpression caveolin-1 was obtained. The results of FCM revealed that the cell cycle of the cells overexpression caveolin-1 arrest at G0/G1 phase(P<0.05), and the transfected cells showed a slower growth rate and formed fewer colonies(P<0.05). overexpression of caveolin-1 slower Hela cells invision ability, migration ability, but quicker the adhersion ability(P<0.05).Conclusion: Overexpression caveolin-1 can restriain the cervical cancer Hela cell line ability of the growth , proliferation , invasion and metastasis. Construction of Caveolin-1RNA interfering eukaryotic expressing vector and its influence on biological behavior of cervical cancer cell line HelaObjective : To design siRNA for caveolin-1(CAV1) and construct eukaryotic expression vector psilence4.1TM-CMV-neo- CAV1,which was transfected into cervical cancer cell line Hela to study the effects of inhibition of CAV1 expression with siRNA on biological behavior of Hela cell line .Methods:Design and chemosynthesize three pairs of double strands siRNA based on the neucleotide sequence of CAV1 gene provided by GeneBank, the siRNAs were transient transfected into Hela, the CAV1 expression of transfected cells were detected respectively by real-time fluorescece quantitation PCR to sieve a valid siRNA sequence. Design and synthesized DNA sequence which can express small hairpin RNA(shRNA), construct RNA interfering eukaryotic expression vector of CAV1 gene, the Hela was stably transfected and was detected the expression of CAV1 gene by RT-PCR, the influence on the cervical cell growth and proliferation after CAV1was inhibited was detected by cell growth curve, clone formation test and FCM.Results : The CAV1 RNA interfering eukaryotic expression vector psilence4.1TM-CMV-neo-CAV1 was successfully constructed, which can significantly reduce the expression of CAV1 after stably transfected to target cells, and significantly influenced the growth and proliferation ablity,invasion and metastasis ability of cervical cancer cells in contrast to the controlled group.Conclusion : The growth and proliferation ablity and invasion and metastasis ability of tumor was remarkably accelerated by inhibitting the expression of CAV1 of cervical cancer cell by RNAi.