Polymerase chain reaction-single strand conformational polymorphism analysis of rearranged during transfection proto-oncogene in Chinese familial hirschsprung's disease

AIM: To investigate the relationship between mutations of rearranged during transfection (RET) proto-oncogene and Chinese patients with Hirschsprung's disease (HD), and to elucidate the genetic mechanism of familial HD patient at the molecular level.METHODS: Genomic DNA was extracted from venous blood of probands and their relatives in two genealogies.Polymerase chain reaction (PCR) products, which were amplified using specific primers (RET, exons 11, 13, 15and 17), were electrophoresed to analyze the single-strand conformational polymorphism (SSCP) patterns. The positive amplified products were sequenced. Forty-eight sporadic HD patients and 30 normal children were screened for mutations of RET proto-oncogene simultaneously.RESULTS: Three cases with HD in one family were found to have a G heterozygous insertion at nucleotide 18 974 in exon 13 of RET cDNA (18 974insG), which resulted in a frameshift mutation. In another family, a heterozygosity for T to G transition at nucleotide 18 888 in the same exon which resulted in a synonymous mutation of Leu at codon 745 was detected in the proband and his father. Eight RET mutations were confirmed in 48 sporadic HD patients.CONCLUSION: Mutations of RET proto-oncogene may play an important role in the pathogenesis of Chinese patients with HD. Detection of mutated RET proto-oncogene carriers may be used for genetic counseling of potential risk for HD in the affected families.

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.

Effect of deleted pancreatic cancer locus 4 gene transfection on biological behaviors of human colorectal carcinoma cells

AIM: To investigate the effect of deleted pancreatic cancer locus 4 (DPC4) gene transfection on biological behaviors of human colorectal carcinoma cells and the role of DPC4 gene in colorectal carcinogenesis. METHODS: PcDNA3.1-DPC4 plasmid was re-constructed by gene-recombination technology. SW620 cells, a human colorectal carcinoma cell line, were transfected with PcDNA3.1-DPC4 plasmid using lipofectamine transfecting technique. Transfected cells were selected with G418. Expression of Smad4 protein was detected in cells transfected with DPC4 gene by immunohistochemistry and Western blot. Biological characterristics of transfected cells were evaluated by population-doubling time and cloning efficiency. Alterations of percentage of S phage cells (S%) and apoptosis rate were determined by flow-cytometry. RESULTS: PcDNA3.1-DPC4 plasmid was constructed successfully. SW620 cells transfected with PcDNA3.1-DPC4 plasmid (DPC4+-SW620 cells) showed a strong intracellular expression of Smad4 protein, and the positive signal was localized in cytoplasm and nuclei, mainly in cytoplasm, where the expressions of Smad4 protein in SW620 cells transfected with PcDNA3.1 plasmid (PcDNA3.1-SW620 cells) and non-transfected SW620 cells (SW620 cells) were weaker than those in DPC4+-SW620 cells. The population-doubling time in DPC4+-SW620 cells (116 h) was significantly longer than that in SW620 cells (31 h) and PcDNA3.1-Sw620 cells (29 h) (P<0.01). The cloning efficiencies of DPC4+-SW620 cells (12%) were markedly lower than those of SW620 cells (69%) and PcDNA3.1-Sw620 cells (67%) (P<0.01). Compared with SW620 cells and PcDNA3.1-Sw620 cells, the Go-G1% of DPC4+-SW620 cells was obviously higher and the S% was markedly lower (P<0.05). Apoptosis rate of DPC4+-SW620 cells was significantly higher than that of SW620 cells and PcDNA3.1-SW620 cells. CONCLUSION: PcDNA3.1-DPC4 plasmid can be successfully re-constructed and stably transfected into human SW620cells, thereby the cells can steadily express Smad4. DPC4 protein may regulate proliferation of colorectal carcinoma cells by inhibiting cell growth and inducing cell apoptosis.

Alterations in metastatic properties of hepatocellular carcinoma cell following H-ras oncogene transfection

AIM: To demonstrate the relationship between H-ras oncogene and hepatocellular carcinoma (HCC) metastasis. METHODS: Activated H-ras oncogene was transfected into SMMC 7721, a cell line derived from human HCC, by calcium phosphate transfection method. Some metastasis-related parameters were detected in vitro, including adhesion assay, migration assay, expression of collagenase IV(c IV ase) and epidermal growth factor receptor (EGFR). RESULTS: The abilities of H-ras-transfected cell clones in adhesion to laminin (LN) or fibronectin (FN), migration, c IV ase secretion increased markedly, and the expression of EGFR elevated moderately. More importantly, these alterations were consistent positively with the expression of p21, the protein product of H-ras oncogene. CONCLUSION: H-ras oncogene could induce the metastatic phenotype of HCC cell in vitro to raise its metastatic potential.

Stable transfection of extrinsic Smac gene enhances apoptosis-inducing effects of chemotherapeutic drugs on gastric cancer cells

AIM: To explore the feasibility of enhancing apoptosis-inducing effects of chemotherapeutic drugs on human gastric cancer cells by stable transfection of extrinsic Smac gene. METHODS: After Smac gene was transferred into gastric cancer cell line MKN-45, subclone cells were obtained by persistent G_(418) selection. Cellular Smac gene expression was determined by RT-PCR and Western blotting. After treatment with mitomycin (MMC) as an apoptotic inducer, in vitro cell growth activities were investigated by trypan blue-staining method and MTT colorimetry. Cell apoptosis and its rates were determined by electronic microscopy, annexin V-FTTC and propidium iodide staining flow cytometry. Cellular caspase-3 protein expression and its activities were assayed by Western blotting and colorimetry. RESULTS: When compared with MKN-45 cells, the selected subclone cell line MKN-45/Smac had significantly higher Smac mRNA (3.12±0.21 vs 0.82±0.14, t=7.52, P＜0.01) and protein levels (4.02±0.24 vs0.98±0.11, t=8.32, P＜0.01). After treatment with 10 μg/mL MMC for 6-24 h, growth inhibition rate of MKN-45/Smac (15.8±1.2-54.8±2.9%) was significantly higher than that of MKN-45 (5.8±0.4-24.0±1.5%, t=6.42, P＜0.01). Partial MKN-45/Smac cancer cells presented characteristic morphological changes of apoptosis under the electronic microscope with an apoptosis rate of 36.4±2.1%, which was significantly higher than that of MKN-45 (15.2±0.8%, t=9.25, P＜0.01). Compared with MKN-45, caspase-3 expression levels in MKN-45/Smac were improved significantly (3.39±0.42 vs0.96±0.14, t=8.63, P＜0.01), while its activities were 3.25 times as many as those of MKN-45 (0.364±0.010 vs0.112±0.007, t=6.34, P＜0.01). CONCLUSION: Stable transfection of extrinsic Smac gene and its over-expression in gastric cancer cell line can significantly enhance cellular caspase-3 expression and activities, ameliorate apoptosis-inducing effects of mitomycin C on cancer cells, which is a novel strategy to improve chemotherapeutic effects on gastric cancer.

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.

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.

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.

Evaluation of transfection methods for transient gene expression in Chinese hamster ovary cells

Three transfection reagents, Lipofectamine&reg 2000, TransIT-PRO&reg and linear 25 kDa polyethylenimine were evaluated for transient expression of enhanced green fluorescent protein in Chinese hamster ovary cells. TransIT-PRO&reg was found to be more efficient under the examined conditions, but comes at an increased cost compared to the widely used PEI.

Effects of Human Insulin Gene Transfection on the Adipogenic Differentiation of Human Umbilical Cord Mesenchymal Stem Cells in Silk Fibroin Scaffolds <i>in Vitro</i>

The resorption of the transplanted fat over time limited the use of autologous fat for the reconstruction of soft tissue defect. Tissue engineering (TE) adipose with silk fibroin scaffold could be a promising substitute for soft tissue filling. In this study, we try to develop a tissue engineering adipose in vitro by seeding silk fibroin scaffold with human umbilical cord mesenchymal stem cells (hUCMSCs) after transfected with recombinant human insulin gene lentivirus. Our aim was to observe the effects of the insulin gene transfection on the adipogenesis of hUCMSCs when cultured with silk fibroin scaffolds. The hUCMSCs infected with recombinant lentiviral pLenti6.3-insulin-IRES-EGFP were seeded on silk fibroin scaffolds and cultured in adipogenic differentiation medium for 5 - 7 days. The expression of adipogenic gene PPARγ-2 was tested by RT-PCR after 7 days culture of adipogenic induction. The accumulation of cytoplasmic droplets of neutral lipids was assessed by Oil Red O staining. The RNA and protein expression of transfected insulin gene in hUCMSCs were detected by QPCR and western blot. The effect of recombinant lentivirus transfection on the growth and proliferation of hUCMSCs was observed by MTT test. We observed that the 2-ΔΔCt value of insulin gene expression of hUCMSCs in the transfected group was 300.25 times higher than that in the untransfected group. The western blot showed that a positive band was discerned at the site of a relative molecular mass of 8 × 103 Dalton in transfected group. After adipogenic culture for 7 days, under the fluorescent inverted phase-contrast microscope, after Oil Red O staining, a lot of adipocytes appeared in silk fibroin scaffold;round adipose droplets showed intracellularly;the size of the adipocytes was not homogenous, and the density of adipocytes in transfected group was significantly higher than that in untransfected group (P = 0.007, P < 0.01). RT-PCR results showed that the expression of adipogenic gene PPARγ-2 in transfected group was much stronger than that in untransfected group. MTT test showed that there was no significant difference in optical density (A) at each time point between transfected group and nontransfected group (P = 0.056, P > 0.05). And there was also no significant difference in optical density (A) between cell group and cell-scalffold group (P = 0.066, P > 0.05). We concluded that insulin gene could obviously promote the adipogenic differentiation of hUCMSCs, and a tissue engineering adipose could be constructed by the silk fibroin scaffolds seeded with human insulin gene-modified hUCMSCs effectively in vitro.

Different Effects of Therapeutic Ultrasound Parameters and Culture Conditions on Gene Transfection Efficiency

Objective： To investigate the effect of different therapeutic ultrasound （TUS） parameters and culture conditions on the cell viability and transfection efficiency of human cervical cancer cells （HeLa）. Methods： HeLa cells were cultured using two different protocols （in suspension or in monolayer）. Subsequently, cells were exposed to different TUS intensity （0.4 W/cm^2, 1.0 W/cm^2, 1.6 W/cm^2, 2.2 W/cm^2）, duty cycle （DC）（10%, 20%, 50%）, exposure time （1 min or 3 min）. Cell viability was analyzed by flow cytometry. Gene transfection of red fluorescent protein （DsRED） was detected. Results： TUS intensity and duty cycle had a great impact on the overall results （P〈0.01）. Cell injury were found to increase progressively with intensity （1.6 W/cm^2, 2.2 W/cm^2） and duty cycle （50%） and cell detachment was accompanied by ultrasound exposure in adherent cells Results of factorial design showed that the fashion of cell culture and the TUS parameters had interaction （P〈0.01）. The ideal conditions that cell viability above 80% producing maximum efficiency were noted to be at 1.0 W/cm^2 irradiated 3 min with a duty cycle of 20% in cell suspension. Conclusion： TUS parameters and transfection conditions have a great impact on the gene transfection and cell viability. Optimal parameters could enhance cell inembrane permeability, which facilitate to delivering the macromolecules into cells.

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.

Effects of Headgroups and Serum on Gene Transfection of Alkaline Amino Acid Based Cationic Lipids

Three cationic lipids with lysylated（1）, histidylated（2）, and arginylated（3） headgroups and cholesterol hydrophobic moiety were synthesized. The average sizes of liposomes and lipoplexes were around 100 and 160 nm, respectively. The gene transfection efficiency of the three lipoplexes loaded with pGL3 or pORF-LacZ was compared on 293T cells in the presence or the absence of serum. The transfection efficiency of the three lipoplexes in a serum-free medium was 2 to 3-fold higher than that of dioleoyl-trimethylammonium propane（DOTAP）. In the presence of serum, however, most of the lipoplexes showed lower transfection activities; only lipoplex 3 retained its high transfection efficiency.

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.

Transfection of Articular Chondrocytes with rhBMP7 Gene and Its Expression

In order to investigate the possibility of expression of exogenous gene in transduced articular chondrocytes, plasmid pcDNA3 rhBMP7 was delivered to cultured chondrocytes. Through immunohistochemical staining and RT PCR assay, the expression of rhBMP7 gene was detected. And the bioactivity of transgene expression product was detected through MTT assay as well. It was confirmed that exogenous gene could be expressed efficiently in transduced chondrocytes and the transgene expression product had obvious bioactivity. The present study provided a theoretical basis for gene therapy on the problems of articular cartilge.

Combined Pluronic P85- and Ultrasound Contrast Agents-mediated Gene Transfection to HepG2 Cells

This study examined the effect of P85 （a pluronic block copolymer） and microbubble （MB） ultrasound contrast agents under ultrasound irradiation on gene transfection and expression. The pEGFP plasmids that can encode enhanced green fluorescent protein （pEGFP） served as a report gene and were mixed with different concentrations of MB/0.05% （w/v） P85. Then the plasmids were transfected into human hepatoma G2 （HepG2） cells. The HepG2 cells treated with MB/P85 or without treatment were exposed to ultrasound （US parameters： 1 MHz, 1.0 W/cm2, 20 s, 20% duty cycle）. Twenty-four hours later, the transfection efficiency was assessed by fluorescence microscopy and fluo-rescence activated cell sorting （FACS） analysis. The cell viability was evaluated by Trypan blue exclusion test. The results showed that the gene transfection efficiency in HepG2 cells under ultrasound irradiation was significantly higher than that without ultrasound irradiation. HepG2 cells in the MB or P85 group in the absence of ultrasound expressed less amount of green fluorescent protein. The expression efficiency reached （22.14±3.06）% and the survival rate was as high as （55.73±3.32）% in the 30% MB plus P85 group. It was concluded that MB and P85 in the presence of ultrasound can enhance gene transfection and expression.