Expression And Significance Of Human Inducible Nitric Oxide Synthase Gene In AIPC Cells

Prostate carcinoma was recognized as a major cancer in the western male population and the second leading cause of male cancer deaths in this population.With living level being improved,there were more and more cases in our country.Surgical treatment,drug treatment, radiotherapy,biotherapy are the current four treatments of disease. although exairesis is the only method of radical cure prostatic carcinoma, 50%patients had metastasis when final diagnosis,Surgical treatment, drug treatment,radiotherapy all can't increase their life span.It is imminent to find new methods.The effect of nitric oxide on many tumors was studied widely.Tumor growth can be promoted by continuous low NO concentration,while cytotoxicity and apoptosis to tumor cells can be induced by quite high NO concentration.For example,mouse melanoma cell and renal cell cancer cell with iNOS gene transfer,can retard tumor development and metastasis.It is a new strategy of curing diseases of cardiovascular system to transfer and recombine NOS gene's by the method of transgene and to re-establish endogenous NO function. Compared with other field,however,the role of nitric oxide in prostate tumor biology was not still understood completely.In early work,we study the effect of exogenous nitric oxide on prostate carcinoma cell,to learn the biological effect of nitric oxide on prostate cancer.We find:exogenous nitric oxide might have a dual biological activity,depending on the concentration of the NO.Lower concentration of NO could promote cell proliferation.On the other hand, higher concentration of NO may cause cytotoxicity via upregulation mRNA and protein of p21^Waf1/ciP1,which resulting in a delay in cell cycle progression and apoptosis.There are many methods to excrete higher concentration of NO such as nitric oxide donor-sodium nitroprusside,but have some defects,for example bad drug tolerance and huge side effect.Our study plans to use mature liposome-induced gene transferring technique currently broadly used in molecular biology labs,we plans to transfect iNOS loss-of-expression human androgen-independent prostatic cancer cell line DU145,obtain the cell line DU145-iNOS which stably expresses iNOS protein,and investigate the biologic effects that derived from iNOS transfection.We wished that we could give a new idea about early diagnoses and therapy on prostate carcinoma.Partâ… Preparation and Identification of Eukaryotic Expression Vector pReceiver-M29-iNOS(ORF)Objective:To amplify and prepare eukaryotic expression vector recombinated plasmid pReceiver-M29-iNOS(ORF),and to perform enzyme-digest identification and DNA sequences analysis,aiming to transfect human androgen-independent prostate cancer line DU 145.Methods:Using restriction endonuclease EcoR I and Xho I enzyme-digest pcDNA3.0-iNOS and pReceiver-M29,Purification and recycling iNOS(ORF) fragment and linearization plasmid pReceiver-M29, connected the products,and then pick monoclonal and shake of small to plasmid and then double-digested PCR testing and detection,when the two results are correct,some transformed frozen glycerol bacteria were performed DNA sequence analysis,outcome were compared with the sequences of iNOS cDNA open reading frame published on internet web by GenBank,analysed by using BLAST biosoftwareResults:We got prospective two fragment bands with sizes respectively comparative to the 6.1 kb vector and 3.3kb iNOS(ORF),after amplified plasmid treated with restriction endonuclease EcoR I,Xho I double-enzyme digest;DNA sequence analysis result has 100% coincidence with the sequences published by GenBank,by BLAST program analysis,reading frame was correct.Conclusion:We amplificated and obtained eukatyotic expression vector plasmid pReceiver-M29-iNOS carrying iNOS cDNA open reading frame full length sequences,reading frame was correct. Partâ…¡Transfection and Screening of Human Inducible Nitric Oxide Synthase Gene in DU145Objective:To prepare human androgen-independent prostate cancer DU145 cell line stably expressing iNOS protein,and then used for subsequent research.Methods:Under 5%CO₂,37℃condition,DU145 cell were incubated in 10%newborn bovine serum DMEM medium.Following cationic liposome Lipofectamine 2000 manual,we transtected DU145 cell with plasmid pReceiver-M29-iNOS and blank vector plasmid pReceiver-M29,cells were devided into such groups as:A group(transfected by pReceiver-M29-iNOS),B group(transfected by pReceiver-M29),B group(the DU145 without transfection.Screening under 400～700μg/ml antibiotic G418 for 2 weeks,next then,cell clones were pick,cultured and propagated under 400～700μg/ml G418 screening pressure.Cellular total RNA of 3 group were drawn out with Trizol method,cDNA was prepared with one-step reverse transcriptional method, and the products of PCR were observed and taken photos after 1% agarose gel electrophoresis;fragments were recovered and to send sequencing,observe under inverted microscope and fluorescence microscope.Results:10 days later,control group DU145 cell were dead,after 14 days,the cells of pReceiver-M29-iNOS group and pReceiver-M29 group started clone-like growth,at 21 day cellular monoclone were pick and cultured for propagation,cDNA was prepared by reverse transcriptional reaction,then PCR reaction,β-actin as the internal control marker, agarose gel electrophoresis,result showed a size-resembling corresponding fagment band in pReceiver-M29-iNOS group,in contrast, no same result in other two groups;under fluorescence microscope,green fluorescent were visible in two transfection group.Conclusion:We successfully established DU145 cellular model stably expressing iNOS protein. Partâ…¢Transfection and Screening of Human Inducible Nitric Oxide Synthase Gene in DU145Objective:To explore the effect of iNOS on the growth, proliferation,apoptosis,discuss the process mechanism of iNOS in prostate cancer.Methods:Under 5%CO₂,37℃condition,DU145 cell were incubated in 10%newborn bovine serum DMEM medium,cells were devided into such groups as:group of transfected by pReceiver-M29-iNOS,group of transfected by pReceiver-M29,group of the DU145 without transfection.We observed the growth and proliferation by MTT method,analyzed the apoptosis by Flow Cytometry method.Results:Under inverted microscope,growth of transfected DU145 cell be came worse,morphological malignant phenotype improved;it's apoptosis enhanced in flowcytometry experiment,MTT test indicated lower and more flat growth curve,slower proliferation velocity of DU145 with transfection(P＜0.05).L-NMMA can enhance its growth,but had no significance(P＞0.05).Conclusion:DU145 with iNOS gene can secrete higher concentration of NO,and NO can induce the growth suppression and enhance apoptosis of DU145 cell in vitro significatively,iNOS gene shows great potential for the gene therapy of androgen-independent carcinoma of prostate.As mentioned above,we transfect iNOS loss-of-expression human androgen-independent prostatic cancer cell line DU145,obtain the cell line DU145-iNOS which stably expresses iNOS protein,we find that DU145 with iNOS gene can secrete higher concentration of NO,and NO can induce the growth suppression and enhance apoptosis of DU145 cell in vitro significatively.But it is not identical in circumstance between cell culture and in vivo,the findings have its limitations.There are so many problems need to solve,we would continue to study,we believe that in near furure there will be substantial breakthrough in gene therapy of gene therapy.