Brain-derived neurotrophic factor genes transfect rat bone marrow mesenchymal stem cells based on cationic polymer vector

BACKGROUND： Gene therapy is an effective expression of genes within target cells after transferring exogenous target genes. Both vector selection and transfection method are important factors for gene transfection. An ideal gene vector is required for a high transfusion of target gene and an exact introduction of target gene into specific target cells so as to express gene products. OBJECTIVE： To study the expression of mRNA and protein after transfecting rat bone marrow mesenchymal stem cells （BMSCs） with brain-derived neurotrophic factor （BDNF） genes based on cationic polymer vector. DESIGN, TIME AND SETTING： A randomized, controlled in vitro study using gene engineering, performed at the Neurobiology Laboratory, Xuzhou Medical College between October 2007 and April 2008. MATERIALS： PcDNA3.1 BDNF was obtained from Youbiai Biotechnological Company, Beijing and cationic polymer vector used was the SofastTM gene transfection reagent that was made by Taiyangma Biotechnological Co., Ltd., Xiamen. METHODS： BMSCs extracted from six Sprague Dawley （SD） rats aged 1 month were isolated and cultured in vitro. Third passage BMSCs were inoculated on a 6-well culture plate at the density of 1×106 cells/L. At about 80% confluence, BMSCs were transfected with PcDNA3.1-BDNF （2 μg） combined with SofastTM gene transfection reagent （6 μg） （BDNF group） or with PcDNA3.1 （2 μg） combined with SofastTM gene transfection reagent （6 μg） （blank vector group）. Cells that were not transfected with any reagents but still cultured under primary culture conditions were used as a non-transfection group. MAIN OUTCOME MEASURES： Enzyme linked immunosorbent assay was used to measure time efficiency of BMSC-secreted BDNF protein. Twenty-four hours after gene transfection, RT-PCR was used to detect expression of BDNF mRNA in the BMSCs. Immunohistochemistry was used to determine expression of BDNF protein in the BMSCs. RESULTS： BDNF protein expression was detected at day 1 after gene transfection, rapidly increased after 5–9 days and gradually increased after 11–15 days in the BDNF group; moreover, BDNF protein expression was higher than that in the non-transfection group and the blank vector group at different time points （P 〈 0.01）. Additionally, BDNF mRNA expression in the BDNF group was higher than that in the blank vector group and the non-transfection group （P 〈 0.01）. CONCLUSION： A cationic polymer vector can effectively mediate the BDNF gene to transfect BMSCs; genetically modified BMSCs can express BDNF protein effectively for a long term.

γ-Ray-Radiation-Scissioned Chitosan as a Gene Carrier and Its Improved in vitro Gene Transfection Performance

Chitosan （CS） is expected to be an ideal gene carrier for its high biosafety. In this work, CS with low molecular weight were prepared through the γ-ray radiation on the acetic acid solution of CS. The CS chains were scissioned under the γ-ray radiation, and the molecu- lar weight （MW） of CS decreased with the absorbed dose. When the absorbed dose was above 30 kGy, the molecular weight of CS decreased about an order of magnitude. The γ-ray-radiation-scissioned CS can effectively bind with plasmid （pEGFP） through complex coacervation method, forming pEGFP/γ-ray-radiation-scissioned CS complex particles with a size of 200-300 nm. The complex particles have good stability and little cytotoxicity. The in vitro gene transfection efficiencies of the pEGFP/γ-ray-radiation-scissioned CS complex particles were investigated by fluorescence microscope and flow cytometry. The results showed that the gene vectors using γ-ray-radiation-scissioned CS as the carrier will possess better gene transfection efficiency than those using natural high-MW CS as the carrier. The higher the absorbed dose, the smaller the MW of CS and the better transfection efficiency of the corresponding gene vector. This work provides a green and simple method on the preparation of CS-based gene vectors with high efficiency and biosafety.

Transfection of the Human Sodium/Iodide Symporter(NIS) Gene with Liposomes and the Expression of the NIS Protein in Human Lung A549 Cancer Cells

OBJECTIVE To examine the possibility of human sodium iodide symporter （hNIS） protein expression in lung cancer cells. METHODS Human lung A549 cancer cells were thawed and cultured in vitro. The cells were divided into an experimental group transfected with a recombinant pcDNA3-hNIS plasmid and a control group transfected only with a pcDNA3 plasmid. The recombinant plasmid vector encoding the hNIS gene （pcDNA3-hNIS） was amplified, purified and identified. The hNIS gene was followed by DNA sequencing. A Western blot and an immunohistochemical assay were applied to detect the hNIS protein expression in the transfected human lung A549 cancer cells. RESULTS Restriction enzyme digestion and DNA sequencing results showed the size and direction of the inserted gene in the recombinant pcD- NA3-hNIS plasmid was correct. The Western blot method and immunohistochemical analysis showed a positive NIS protein expression in the experimental group. The NIS protein was detected mainly in the cell membranes showing a positive rate up to 70.6% with no expression of the NIS protein in the control group. There was a significant difference between two groups （P=0.000）. CONCLUSION The hNIS gene was transfected effectively into human lung A549 cancer cells mediated by Lipofectamine 2000, and was expressed with its protein in vitro.

Granulocyte-macrophage colony-stimulating factor-transfected bone marrow stromal cells for the treatment of ischemic stroke

Adult, male, Sprague-Dawley rats were injected with granulocyte-macrophage colony-stimulating factor-transfected bone marrow stromal cells （GM-CSF-BMSCs） into the ischemic boundary zone at 24 hours after onset of middle cerebral artery occlusion. Results showed reduced infarct volume, decreased number of apoptotic cells, improved neurological functions, increased angiogenic factor expression, and increased vascular density in the ischemic boundary zone in rats that underwent GM-CSF-BMSCs transplantation compared with the BMSCs group. Experimental findings suggested that GM-CSF-BMSCs could serve as a potential therapeutic strategy for ischemic stroke and are superior to BMSCs alone.

Anticancer Drug Resistance of HeLa Cells Transfected With Rat Glutathione S-transferase pi Gene

To establish a cytologic expressing system of rat glutathione S-transferase pi (GST-pi) cDNA for detecting the resistance of HeLa cells to anticancer drugs. Methods The assessment was made with various anticancer drugs (adriamycin, mitomycin, cisplatinum and vincristine) that showed different cytotoxicities in transfectant HeLa cells with pSV-GT containing rat GST-pi cDNA (HeLa/pSV-GT) or control pSV-neo (HeLa/pSV-neo). Expression levels of GST-pi mRNA in HeLa/pSV-GT and HeLa/pSV-neo were measured by in situ hybridization using Digoxin-labelled cDNA probe. Results HeLa/pSV-GT expressed significantly high degree of GST-pi mRNA, whereas both HeLa/pSV-neo and HeLa cells had very low expression. Cytotoxicities of HeLa/pSV-GT and HeLa/pSV-neo with 4 anticancer drugs were measured by MTT assay. Drug concentrations for yielding 50% inhibition (IC50) in HeLa/pSV-GT by adriamycin, mitomycin and cisplatinum were 70.13 靏/mL, 10.95 靏/mL and 16.52 靏/mL, respectively. In contrast, IC50 in HeLa/pSV-neo was 10.34 靏/mL, 7.48 靏/mL and 13.70 靏/mL, respectively. The cytotoxicities of vincristine on both HeLa/pSV-GT and HeLa/pSV-neo were not significantly different. Conclusions Our findings suggest that HeLa/pSV-GT containing rat GST-pi cDNA is resistant to some anticancer drugs due to overexpression of GST-pi. Also, HeLa/pSV-GT cell line could serve as a useful cytogenetic model for further research.

BASIC FIBROBLAST GROWTH FACTOR GENE TRANSFECTION TO ENHANCE THE REPAIR OF AVASCULAR NECROSIS OFTHE FEMORAL HEAD

Objective To explore a new method for the therapy of avascular necrosis of the femoral head. Method The recombinant plasmid pCD-rbFGF was mixed with collagen and was implanted in the necrotic femoral head. Expression of basic fibroblast growth factor (bFGF) was examined by RT-PCR and immunohistochemical method. Re-pair of the femoral head was observed by histological and histomorphometric analysis. Result Expression of bFGF was detected in the femoral head transfected with bFGF gene, indicating significant increase of angiogenesis 2 weeks after gene transfection and increased new bone formation 8 weeks after gene transfection on histom-orphometric analysis (P< 0.01). Conclusion Transfection of bFGF gene enhances bone tissue angiogenesis. Repair in osteonecrosis would be accelerated accordingly.

Ultrasound-targeted cationic microbubble-mediated gene transfection and inhibition of retinal neovascularization

AIM:To investigate whether ultrasound-targeted cationic microbubbles(CMBs)destruction could deliver endostatingreen fluorescent protein(GFP)plasmids efficiently to the human retinal endothelial cells(HRECs)and inhibit retinal neovascularization in mice.METHODS:CMBs were prepared and the presentation of GFP reporter was confirmed by flow cytometry and laser confocal microscopy.Experiments assessing HRECs migration and vascular formation were per formed to evaluate gene therapy’s efficiency in vitro.A mouse model of oxygen-induced retinopathy was employed and the expression of Bcl-xl,Bcl-2,vascular endothelial growth factor(VEGF)and endostatin in the retina of mice were determined by Western blotting and quantitative polymerase chain reaction(q PCR).The expression of endostatin-GFP in the retina was examined by laser confocal microscopy at 5,14,and 28 d after treatment.RESULTS:The gene expression of endostatin was the highest in the group of the CMBs.Besides,the inhibition and antiangiogenesis effect of the migration and development of HRECs were improved following treatment with CMBs compared with the other groups in vitro.In vivo,retinal neovascularization was significantly inhibited and the fluorescence intensity of endostatin-GFP in the mouse retina was importantly higher in the group of CMBs than that in other groups.CONCLUSION:The research illustrates ultrasoundtargeted CMBs destruction possessed distinct effect on the inhibition of the vascular formation and the development of retinal neovascularization both in vitro and in vivo.

A study of the expression of p53 in posttransfection cells with rAdp53 gene and inhibitory activity in vitro

Objective： To investigate the inhibitory effect and IC50, （rAdp53） in colorectal cancer cells in vitro and to guide （50% inhibiting concentration） of the recombinant adenoviral p53 gene clinical practice. Methods： We evaluated the efficiency （IC50）of the rAdp53 and six kinds of anti-cancer drugs（5-fluorouracil, tegafur, mitomycin c, cisplatin, oxaliplatin, paclitaxel） in human colorectal cancer cell line-174 through the cell culture and MTT chemosensitivity assay to make sure the anti-cancer capability of rAdp53. Expression of p53 protein in transfection cells of colorectal cancer line-174 with rAdp53 was evaluated by immunohistochemical staining. Results： The rAdp53 is a dose-and time-dependent anti-cancer drug, its IC50 is 5.73×10^11 VP/ml, but its effect was not obvious when compared with other anti-cancer drugs. In control group, the immunohistochemistry stain was negative. However, rAd-p53 of five different concentrations were all positive in infected colorectal cancer cells with rAd-p53 and the earliest positive result would present 24 hours after infection. Conclusion： The rAdp53 has good anti-cancer efficacy is colorectal cancer cell line-174 in vitro. But its anti-cancer efficacy was less than those of the classical chemical medicine mitomycin c, 5-fluorouracil and cisplatin etc., when it was used alone.

Hydrodynamics based transfection in normal and fibrotic rats

AIM： Hydrodynamics based transfection （HBT）, the injection of a large volume of naked plasmid DNA in a short time is a relatively simple, efficient and safe method for in vivo transfection of liver cells. Though used for quite some time, the mechanism of gene transfection has not yet been elucidated. METHODS： A lucJferase encoding plasmid was injected using the hydrodynamics based procedure into normal and thioacetamide-induced fibrotic Sprague Dawley rats. Scanning and transmission electron microscopy images were taken. The consequence of a dual injection of Ringer solution and luciferase pDNA was followed. Halofuginone, an anti collagen type I inhibitor was used to reduce ECM load in fibrotic rats prior to the hydrodynamic injection. RESULTS： Large endothelial gaps formed as soon as 10＇ following hydrodynamic injection; these gradually returned to normal 10 d post injection. Hydrodynamic administration of Ringer 10 or 30 m prior to moderate injection of plasmid did not result in efficient transfection suggesting that endothelial gaps by themselves are not sufficient for gene expression. Gene transfection following hydrodynamic injection in thioacetamide induced fibrotic rats was diminished coinciding with the level of fibrosis. Halofuginone, a specific collagen type I inhibitor, alleviated this effect. CONCLUSION： The hydrodynamic pressure formed following HBT results in the formation of large endothelial gaps. These gaps, though important in the transfer of DNA molecules from the blood to the space of Disse are not enough to provide the appropriate conditions for hepatocyte transfection. Hydrodynamics based injection is applicable in fibrotic rats provided that ECM load is reduced.

Brain-derived neurotrophic factor gene transfection promotes neuronal repair and neurite regeneration after diffuse axonal injury

This study sought to assess the potential of brain-derived neurotrophic factor （BDNF） to promote neuronal repair and regeneration in rats with diffuse axonal injury, and to examine the accompanying neurobiological changes. BDNF gene transfection reduced the severity of the pathological changes associated with diffuse axonal injury in cortical neurons of the frontal lobe and increased neurofilament protein expression. These findings demonstrate that BDNF can effectively promote neuronal repair and neurite regeneration after diffuse axonal injury.

Protection of rat islet viability following heme oxygenase-1 gene transfection via adenoviral vector in vitro

Objective： To investigate the effect of Heme oxygenase-1 （HO-1） gene transfection on the viability of cultured rat islets, and to explore the potential value of HO-1 gene in islet transplantation. Methods：Recombinant adenovirus vector containing human HO-1 gene（Ad-HO-1 ） or enhanced green fluorescent protein gene（Ad-EGFP） was generated by using AdEasy system respectively. The rat islets were transfected with Ad-HO-1, Ad-EGFP or blank vector and then cultured for 7 days. Transfection was confirmed by expression of EGFP and human HO-1 protein detected by fluorescence photographs and western blot, respectively. The insulin release upon different concentration of glucose stimulation was detected using insulin radioimmunoassay kit, and stimulation index（SI） was calculated. Glucose-stimulated insulin release was used ＇to assess islet viability. Results：Adenovirus vector successfully transferred HO-1 gene to rat islet cells in vitro, and the insulin release upon high level of glucose stimulation and stimulation index （SI） of Ad-HO-1-infected islets were significantly higher than those of Ad-EGFP-infected islets and control islets （P 〈 0.05）. Conclusion： Adenovirus-mediated HO-1 gene transfection is a feasible strategy to confer cytoprotection and therefore protect the viability of cultured rat islets.

THE EFFECTOR FUNCTIONS OF MACROPHAGES TRANSFECTED WITH INTERFERON-GAMMA GENE MEDIATED BY RECOMBINANT ADENOVIRUS

Macrophages(Mφs) are not only a kind of immune effector cells but also a kind of antigenpresenting cells(APC). In order to improve their antitumor effect, we transfected interferongamma(IFNγ) gene into Mφs by recombinant adenovirus because IFNγis a kind of potent macrophageactivating factor(MAF). High level of IFNγ could be detected in the supernatants of Mφs after IFNγ gene transfection and IFNγ secretion peaked at 20 hour. The cytotoxicity of IFNγgenetransfected Mφs increased significantly. The secretion of TNF, IL1, nitric oxide(NO) also increased to some extent. The results demonstrated that recombinant adenovirusmediated IFNγ gene transfection could improve the effector functions of Mφs efficiently.

THE EFFECT OF TRANSFECTED CX43 GENE ON THE GJIC AND PROLIFERATION OF GLIOMA CELLS

Objective: To evaluate the effect of Cx43 gene on gap junction intercellular communication (GJIC) and proliferation of glioma cells. Methods: Cx43 cDNA was transfected into TJ905 human glioblastoma cells using lipofectamine. The expression of Cx43 was identified by Northern blot analyses, in situ hybridization and immunohistochemistry. MTT assay and average number of AgNORs (Argyrophlic nuclear organizer regions) were used to determine the cell proliferation. TUNEL method was used for detection of cell apoptosis, and scrape loading and dye tranfer method for examination of GJIC. Results: The Cx43 expression was greatly upregulated when Cx43 gene was transfected into TJ905 glioma cells. The cell proliferation was inhibited while the cell apoptosis was not increased and GJIC was significantly restored in the glioma cells transfected with Cx43 gene. Conclusion: Cx43 gene has an inhibitory effect on the glioma cell proliferation, but no effect on induction of cell apoptosis. The restoration of GJIC may be the major mechanism involved in its effect. Cx43 gene can be the candidate for gene therapy of gliomas.

EFFECTS OF CARBOXYMETHLY DEXTRAN MAGNETIC NANOPARTICLES CARRIER SYSTEM ASSOCIATED WITH EXTERNAL MAGNETIC FIELDS ON KILLING TUMOR CELLS AND GENE TRANSFECTION

Objective: To investigate the preparation of the carboxymethly dextran iron oxide magnetic nanoparticles (CDMN) and the effects of CDMN carrier system associated with external magnetic fields on killing tumor cells and gene transfection in vitro. Methods: Epirubicin-CDMN (EPI-CDMN) and green fluorescent protein (GFP) plasmid-CDMN (GFP-CDMN) were prepared by the oxidation-reduction procedure and their characters were detected, respectively. The effects of EPI-CDMN associated with external pulsed electromagnetic fields (PEMFs) (10 mT) on killing human bladder cancer BIU-87 cells were studied by MTT assay and Annexin-V/PI double-labeled flow cytometry technique, respectively. And the transfection efficiency of GFP when CDMN were used as gene carrier associated with the external magnetic fields was evaluated under fluorescence microscope in vitro. Results: The diameter of EPI-CDMN and GFP-CDMN were about 8~10 nm and 5~9 nm, respectively, and saturation magnetization were 0.22 emu/g and 0.26 emu/g, respectively. EPI-CDMN associated with PEMFs could significantly inhibit the proliferation of BIU-87 cells and induce cells apoptosis, the growth inhibitory rate and apoptosis rate were (21.82±3.18)% and (16.79±3.37)%, respectively. The transfection efficiency of GFP-CDMN combined with PEMFs was significant higher than that of GFP-CDMN without PEMFs [(45.70±4.32)% vs (35.85±2.16)%, P<0.05]. Conclusion: It seemed that EPI-CDMN associated with external magnetic fields could significantly killed human bladder cancer BIU-87 cells and CDMN could effectively transfer GFP gene into tumors cells with the help of external magnetic fields which provided experimental basis for the magnetic targeting therapy of tumor.

Establishment of hepatocarcinoma cell line transfected by the B7 gene and its biocharacteristics

OBJECTIVE: To study the function of costimulation sign in tumor immunology and construct the new cell line B7^+ Smmc7721. METHODS: The B7 gene was transfected into the hepatocarcinoma cell Smmc7721 by liposma. Polymerase chain reaction (PCR) and reverse transcriptase-PCR (RT-PCR) were applied to test the result. MTT colorimetric assay was used to value the killing effect of lymphokine activated killer cells (LAK) activated by IL-2 on the transfected cell line and the original line. RESULT: B7^+ Smmc7721 was improved to be steadily expressed B7 molecule and LAK cells could more effectively act on the B7^+ Smmc7721 cells. CONCLUSION: The B7 gene can be transfected to hepatocarcinoma cells and can be expressed steadily in vitro, thus increasing the efficiency of LAK cells activated by IL-2 on them.

Editor's Choice—Adenovirus-mediated gene transfection of stem cells

Neural stem cells transplantation plays an important role in repair and cell replacement therapy for nervous system degenerative diseases. However, the self-repair effects are minimal because of a limited absolute number, as well as the local microenvironment, post-transplantation. A combined treatment utilizing stem cell transplantation and gone therapy can exert a dual effect involving stem cells and neurotrophic factors. The adenovirus carrier is

Gene Transfection Mediated by Ultrasound and Pluronic P85 in HepG2 Cells

In order to assess whether gene transfection could be mediated by ultrasound in association with P85 and find the appropriate parameters of ultrasound irradiation, the effects of ultrasound with or without P85 on gene transfection of HepG2 cells were examined. The HepG2 cells were irra- diated by ultrasound at 1 MHz, 0.4-2.0 W/cm2 and 50% duty cycle with plasmid encoding enhanced green fluorescent protein （EGFP） as a report gene. Forty-eight h later, the expression of EGFP was detected under the fluorescence microscopy. Transfection efficacy was quantitatively assessed by flow cytometry, and cell viability was evaluated by trypan blue exclusion. The results showed that the transfection efficacy was increased with the increases in ultrasound output power and the ideal transfection efficacy was achieved in HepG2 cells irradiated by ultrasound at 0.8 W/cm2 for 30 s. The transfection efficacy in ulstrasound＋P85 group was three times higher than in single ultrasound group [（17.63±1.07）% vs （5.57±0.56）%, P〈0.05]. The cell viability was about 81% and 62% in ultrasound group and ultrasound＋P85 group respectively. It was concluded that ultrasound in combination with P85 could mediate the gene transfection of HepG2 cells, ideal transfection efficacy was achieved by ultrasound irradiation at 0.8 W/cm2 for 30 s, and P85 could somewhat increase the damage to cells caused by ultrasound.

Cationic Liposome-mediated bcl-xl Gene Transfection into Human Keratocytes

The efficiency and safe range of LipofectamineTM2000 (LF2000)/bcl-xl applied in human keratocytes, the optimal ratio of LF2000/bcl-xl and the bcl-xl gene expression in human keratocytes were investiaged. By using trypan-blue staining, the effects of LF2000 and bcl-xl on the survival rate of the cultured human keratocytes were measured respectively. By using semi-quantitative RT-PCR, the efficiency and the expression of LF2000-mediated bcl-xl transfection into keratocytes were examined. The results showed that the survival rate of human keratocytes had no signficant change in the presence of LF2000 (20 μg/ml) or bcl-xl (10 μg/ml) for 24 h. LF2000 could effectively mediate the transfection of exogenous gene bcl-x1 into human keratocytes. The best transfection efficiency could be obtained when the ratio of bcl-xl/LF2000 was 1:8. One day after transfection, the positive cells for bcl-x1 could be detectable, and the positive rate reached the peak on the post-transfection day 3 (48.3 %), then gradually decreased. Fifteen days after transfection, there were few positive cells. It was suggested that LF2000 could effectively transfer the exogenous gene bcl-xl into human keratocytes without obvious toxicity during a concentration range. LF2000/bcl-xl may be likely to play an important role in gene therapy of human keratocytes.

Effects of Transfection of ICAP-1α and Its Mutants on Adhesion and Migration of 2H-11 Cells

This study examined the effect of integrin cytoplasmic domain-associated protein 1α (ICAP-1α) and its mutatants T38A and I138A on the adhesion, migration and tube formation of 2H-11 cells.rAAV-ICAP-1α, rAAV-T38A and rAAV-I138A were constructed.After infection, the expression of ICAP-1α and p-ERK1/2, p-c-Jun protein was measured by Western blotting.Adhesion ability was evaluated by using MTT.Cell migration was determined by using Boyden chamber method.Tube formation test was conducted on Matrigel.The results showed that in ICAP-1α, T38A and I138A groups, ICAP-1α protein expression was increased.In T38A and I138A groups, phospho-ERK1/2, phospho-c-Jun protein expressions were significantly increased as compared with the control group and the GFP group.ICAP-1α group protein expression was obviously decreased when compared with the control group and the GFP group.Cell adhesion ratio was 0.1429±0.0080 in control group, 0.1434±0.0077 in GFP group and the ratio in T38A and I138A groups increased to 0.3210±0.0082 and 0.3250±0.0079, respectively.In ICAP-1α group, the ratio was decreased to 0.1005±0.0073.In T38A and I138A groups, the number of migrating 2H-11 cells was increased to 31.45±3.20 and 33.10±5.40 against 18.51±2.80 in control group and 20.47±3.12 in GFP group.In ICAP-1α group, the number was decreased to 12.06±1.72.The number of tube-like structures was increased to 20.41±2.54 in T38A and to 22.26±3.07 in I138A groups as compared to those of control group 12.45±1.84 and GFP group 13.63±2.71.In ICAP-1α group, the number of tube-like structures was decreased to 8.32±1.24.It was suggested that rAAV-T38A and rAAV-I138A transfection can substantially increase 2H-11 cell adhesion, migration and angiogenisis, while rAAV-ICAP-1α can greatly inhibit the effect.These effects might be correlated with ERK1/2 and c-Jun protein phosphorylation.

Expression of Vascular Endothelial Growth Factor (VEGF) in Human Osteosarcoma Cells Transfected with Adeno-associated Virus-antisense VEGF

Summary: The expression of protein vascular endothelial growth factor (VEGF) in osteosarcoma cells transfected with adeno-associated virus (rAAV)-antisense VEGF was studied to provide the foundation of osteosarcoma treatment through antivascularization. The rAAV-antisense VEGF at different doses (0, 20, 50, 100, 200, 240 μl) was transfected into osteosarcoma MG-63 cell. The cells and culture supernatants were collected before and after tansfection. The expression of VEGF protein was detected by using immunohistochemical staining (SP) and Western blot. SP and Western-blot tests revealed that the MG-63 Cells transfected with rAAV-antisense VEGF had less staining than those without transfection with rAAV-antisense VEGF, and the staining intensity was negatively correlated with the doses of genes. The corresponding A values of transfected genes with different doses of rAAV-antisense VEGF (0, 20, 50, 100, 200, 240 μl) were 86 614±13 776, 73 245±15 414, 61 078±12 124, 54 657±10 953, 39 802±11 308, 32 014±15 057 respectively, with the difference being significant (P<0.05). It was concluded that the expression of VEGF protein in MG-63 cells could be inhibited by rAAV-antisense VEGF.