| Background: Colon carcinoma is a common malignant tumor. It hasshowed that morbility of colon carcinoma and rectal carcinoma is thethird of malignant tumor, and mortality is the forth. It became more andmore obvious that the tendency of morbility of cancinoma of largeintestine is increasing recently, annual incremental rate of morbility ofcolon carcinoma is 4％in cities, while the number of male patientsincreased up to 58.8％. Most patient are in middle-advanced stage whenthey visit the doctor and it appears transfer or high recurrent afteroperation because it is latent, short of specificity and difficult earlydiscovery. Chemotherapeutics has severe side effects,such as bonemarrow depression, damage of heart,liver and kidney. Exairesis isimportant therapeutic tool, but it leads to toxicity of chemo-therapy, shiftand high recurrent after operation for moderate advanced patients.So it isnecessary to search for a new approach. Gene therapy is hot study, but itconfines on safety of carrier and poor target.The key of gene therapy is vector. Viral vector and non-viral vectorhad wide application. Viral vector mainly contain adenovirus, retrovirus,adeno-associated virus and so on, but it has potential danger of safety; it isrepeled by immune system when it is injected to organism for a greatimmunogenicity. An injection with adenovirus vector with highconcentration, it leads to serious inflammatory reaction of liver. Viralvector such as liposome and polycation are commonly used lately. But,liposome and polycation have low specificness and targetness of genetransfer tissue, have lower transfection efficiency and short period ofgene expression, for they can be phagocytized by endothelial system. Therefore, exploration for a new non-viral vector is a hot spotNanoparticles has a great chemical activity because of small dimensioneffect and surface effect. What's more, its specific surface is increasedlargely with shrink of diameter. So nanoparticles becomes non-viralvector with remarkable prospect. It is reported that transit time ofnanoparticles in vivo is longer than that of particles with normal size. Itcan't be removed by phagocyte in a short period for the former, thenmuch more exud to outside of blood vessel, tissue space, lengtheningcontact time of cells,and improving transfection efficienty. Plasmid DNAis digested and degradated by nuclease when it enters the vessel.nanoparticles genetic carrier can condense and protect DNA, formingcompact structure with DNA avoid of degradation of nuclease.Many approaches such as transcribe technology with targetnesshave been worked out in order to improve the targetness vector of genetherapy and to decrease damage of normal cells, transcribe technologywith targetness utilize specific promotor of tissue and cell to controltranscrip-tion of gene, then therapy gene can be expressed in specificcells. CEA is relatively specific marker of maliganant tumors such ascolon cancer, whose masculine rate in colon carcinoma is 70％-90％, andshift and recurrent rate is still higher. So it can be essential momitoringindex of colon carcinoma and its tumor specificness is apply by manygene therapy of tumor. Transcriptional regulatory element of CEAcontains enhancer element and promotor in the 5th flanking region whichcan control expression of objective gene in positive cancer tissue of CEA.Objectives: To explore the joint promoter with high activity,consisting CEA promotor and CMV enhanser. To improve sepcificness ofexpression of suicide gene yCDglyTK in target cells under control of fusepromoter. Calcium phosphate nanoparticles vector with goodbiocompatibility and have no toxicity. Suicide gene is introducted into cells of colon carcinoma by nanoparticles, for purpose to enhance genetherapy targetness and security.Methods: We prepared a kind of calcium phosphate nanoparticleswith 30nm and uniform dissipation by method of chemistry, and constructthe expression plasmid vector of pcDNA3.1(-)CVyCDglyTK controlledunder CEA promoter and cytomegalovirus(CMV) enhancer with PCR,PT-PCR,confusion PCR,enzyme restriction, ligation; Plasmid expressionvector pcDNA3.1(-)CV·yCDglyTK was transfection lovo cell line ofcolon carcinoma by nanoparticles in vitro. Detect the efficiency oftransfection by fluorescence microscope and flow cytometry, Appraiseexpression of mixed suicide gene of yCDglyTK in lovo cell line bymethod of RT-PCR and Western-blot. Detect toxicity of 5-FC and GCV towild type lovo by MTT. detect lethal effect of suicide prodrug system tolovo cell by MTT and clone formation experiment. Draw growth curve ofcell to show the influence on growth of lovo cell. Contrast the expressionlevels of suicide gene before and after therapy by way of immunitychemistry and immunofluorescence. Determine the change of prodrug5-FC to 5-FU before and after treatment by HPLC. Positive clone cell ofpcDNA3.1(-)CV·yCDglyTK transplant Balb/C nude mouse to carry outexperiment of tumor therapy in vivo.Results: Calcium phosphate nanoparticles can binds with plasmidDNA after it is modified by calcium chloride with different concentration.The complex adsorb cell envelope and enters cells, increa-seing capacityof DNA in cell, boosting efficiency of gene transcripttion, resistingdegradation of DNA and protecting integrity of DNA. the efficiency oftransfection to achieve 53.38％detect by flow cytometry, We discoveredthat 5-FC and GCV had lethal effect on wild type lovo cell when they arein high concentration(5-FC＞200ug/ml, GCV＞50 ug/ml).The rate of cellsurvival was 63.23％and 11.02％respecti-vely, when their concentration were 20ug/ml and 200ug/ml. Moreover, when two kinds of prodrug, 5-FCand GCV were simultaneously existing, positive lovo cell ofyCDglyTK had no coordinated killing tumor which was different frommuch report, and its action was lower than that of being used 5-FCalone. Concentration of 5-FU was 168ug/ml and transform efficiencywas 84％by HPLC when positive lovo cell of yCDglyTK was handled by5-FC for 24 hrs. It indicated from side killing effect that the transfectionefficiency ofplasmid pcDNA3.1(-)CV·.yCDglyTK was 20％when 5-FCwith concentration of 200 ug/ml was present, and relative survival rate ofcell would decrease to 32.6％, So the mixed suicide gene prodrug systemhad powerful side killing effect. From the animal experiment, comparedwith lovo+5-FC group, CPNP-CVyCDglyTK+5-FC group, CPNP-CMVyCDglyTK+5-FC,and yCDglyTK+PBS group, there were greatdifferences in tumors for yCDglyTK+5-FC group (P＜0.05).So thesuicide gene prodrug therapy mediated by nanoparticles showed obviousaction of restraining tumor.Conclusions:1) calcium phosphate nanoparticles can effectivelymediate pcDNA3.(-)CV·yCDglyTK suicide gene plasmid to transfectlovo cell, CPNP had no obvious cytotoxicity, so it's may be a novelnon-viral vector in gene therapy.2) 5-FC and GCV had obvious lethal effect on yCDglyTK positivecells, it had no coordinated actions and its action was lower than that of5-FC while higher than that of GCV.3) The mixed suicide gene prodrug system had powerful side killingeffect.4) For mixed suicide gene prodrug system,the concentration of 5-FCwas lower than 200 ug/ml, GCV lower than 50 ug/ml, which had lowercytotoxicity to lovo cell having no suicide gene in vitro.5) calcium phosphate nanoparticles mediated Suicide gene controlled by CEA had good targetness in tumor therapy both in vivo andvitro.
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