The Research Of Anti-Colon Carcinoma Effect Of The Therapic System PcDNA3.1(-)shVEGF/yCDglyTK

Background and Aim:Colon carcinoma is one of most common malignancies, which is the fourth most common cancer in men and the third in women worldwide. In China, the incidence is in the fourth place of all malignancies. Nowadays the chief therapy of colon carcinoma is surgery, coupled with radiotherapy and chemotherapy. A majority of patients has been found in the advanced stage and loses the chance of surgery because the symptoms are occult. Furthermore, radiotherapy and chemotherapy have a severe toxic and side-effect and weak specificity. Consequently, the gene targeted therapy of colon carcinoma has become the focus of researches. The vectors of gene therapy consist of two kinds, viral vectors and non-viral vectors. Because of immunogenicity, viral vectors can induce immunological reaction of body and have potential risk of safety, while non-viral vectors are non-toxic and safe, which are applied more in researches. As a kind of non-viral vectors, the calcium phosphate nanoparticle (CPNP) has many nice characteristics like high transfection efficiency, nonpoisonous side effect to human body, and high safety and so forth. The suicide genes such as CD/5-FC and HSV-TK/GCV systems have a lethal effect on colon carcinoma cells. Furthermore, the synergistic action of the unity of CD and TK has a more powerful lethal effect on carcinoma cells than each of them. Tumor angiogenesis plays a vital role in tumor growth and metastasis. Vascular endothelial growth factor (VEGF) is one of most important substances to promote tumor angiogenesis. RNA interference can efficiently inhibit tumor angiogenesis by highly specific silence of VEGF gene expression, and eventually lead to the purpose of tumor growth inhibition. Hence, in this experiment, the non-virus vector CPNP is used as a gene transfection vector, and the integration of cytomegalovirus (CMV) enhancer, carcinoembryonic antigen (CEA) promoter and fusion suicide gene yCDglyTK combined with RNA interference of VEGF has a targeted lethal effect on human colon cancer cells Lovo in vitro. Method:The Lovo cells were divided into five group, one of which was the blank controller (Group L1), and the rest were separately transiently transfected into pcDNA3.1 (-) Null (Group L2),pcDNA3.1 (-) CVyCDglyTK (Group L3),pGenesil-shVEGF (Group L4),pcDNA3.1(-)-shVEGF/yCDglyTK(Group L5) using CPNP as a vector. The expression of the yCDglyTK and VEGF gene was detected by RT-PCR and immunofluorescence. MTT assay and flow cytometry (FCM) were used to detect the cytotoxic effects and apoptosis rates of the yCDglyTK/ 5-FC system. SPSS 13.0 and Excel 2003 were used in data analysis. There was a statistical significance when P＜0.05.Results:1. The expression of yCDglyTK and VEGF genes:(1) The expression of yCDglyTK mRNA and protein was enhanced in Group L3 and L5; (2) All groups expressed VEGF mRNA and protein, but the expression was weakened in Group L4 and L5. It showed that CPNP can successfully induce the transfection of four plasmids into Lovo cells.2. The anti-proliferative effect of transfection of CPNP-DNA in Lovo cells:MTT analysis showed that Group L5 had the lowest survival rate of all on the effect of 5-FC (P<0.05). It showed that transfection of the united gene therapic system pcDNA3.1(-)shVEGF/yCDglyTK had a more potential anti-proliferative effect on Lovo cells than yCDglyTK or shVEGF separately.3. The apoptosis of transfection of CPNP-DNA in Lovo cells:The apoptosis rates of FCM were as follows:Group L1 1.92%, Group L2 5.57%, Group L3 43.8%, Group L4 20.2%, Group L5 67.8%. Group L5 had the highest apoptoticrate. It showed that the transfection of the united gene therapic system pcDNA3.1(-)shVEGF/yCDglyTK led to higher apoptosis in Lovo cells than yCDglyTK or shVEGF separately.Conclusions:1.CPNP can successfully induce the transfection of pcDNA3.1(-)Null, pcDNA3.1(-)CVyCDglyTK, pGenesil-shVEGF and pcDNA3.1(-)shVEGF/yCDglyTK into colon cancer Lovo cells.2. The yCDglyTK gene can effeciently inhibit the proliferation of Lovo cells, and VEGF-shRNA can efficiently inhibit the expression of VEGF.3. The united gene therapic system of pcDNA3.1(-)shVEGF/yCD -glyTK is much more efficienct than suicide gene therapy or RNA interference separately in killing colon cancer Lovo cells.