In the present study, packaging system composed of pAAV-CMV-GFP, pAAV-RC and pHelper were transfected into human embryonic kidney 293 cells (HEK293 cells) mediated by polyethyleneimine (PEI) to explore an optimal transfection condition. Different total plasmid DNA dosages (1, 2, 3, 4, 5, 6 μg) and different PEI/Plasmid ratios (1:1, 3:1, 5:1, 7:1) were tested with detection of green fluorescence protein (GFP) with ImagePro Plus6. 0 Software. Then transfection efficiency of the optimized transfection system was further observed for different time periods(12, 24, 36, 48, 60, 72 h). The results showed that total plasmid dosage of 4 μg/well with PEI/plasmid ratio of 3 : 1-5 : 1 was an efficient transfection condition. Transfection efficiency-time curve was an S-shaped curve. Transfection efficiency reached a plateau at 60 h after transfection. The optimized conditions for PEI-mediated transfection at the optimal time result in enhanced transfection efficiency of triple plasmid into HEK293 cells.
Green Fluorescent Proteins ; HEK293 Cells ; Humans ; Plasmids ; Polyethyleneimine ; Transfection ; methods

Apoptosis is a physiologically essential mechanism of cell and plays an important role in reducing the development and progression of tumors. The appealing strategy for cancer therapy is to target the lesions that induce apoptosis in cancer cells. Survivin, the smallest member of the mammalian inhibitors of the apoptosis protein family, is upregulated in various malignancies to protect cells from apoptosis. Survivin knockdown could induce cancer cell apoptosis and inhibit tumor-angiogenesis. Survivin expression would be silenced by microRNA (miRNA)-mediated RNA interference. However, noninvasive and tissue-specific gene delivery techniques remain absent recently and the utilizations of miRNA expression vectors have been limited by inefficient delivery technique, especially in vivo. On the other hand, safe and promising technologies of gene transfection would be valuable in clinical gene therapy. Successful treatment of gene transfer method would lead to a new and readily available approach in the anticancer research. Sonoporation is an alternative technique of gene delivery that uses ultrasound targeted microbubble destruction to create pores in the cell membrane. Based on our previous studies, in this article, we postulated that the transfection of miRNA could be mediated by the combination of sonoporation and polyethylenimine (PEI) which was one of the most effective poly-cationic gene vectors and enhance the endocytosis of plasmids DNA and hypothesized that the gene silencing and apoptosis induction with miRNA targeting human Survivin would be improved by this novel technique. In our opinion, this novel combination of sonoporation and PEI could enhance targeted gene delivery effectively and might be a feasible, novel candidate for gene therapy.
Genetic Therapy ; methods ; Humans ; Inhibitor of Apoptosis Proteins ; genetics ; MicroRNAs ; genetics ; Neoplasms ; therapy ; Polyethyleneimine ; chemistry ; Transfection ; methods

Polyethylenimine (PEI) is a kind of nanometer nonviral vector frequently applied in gene transfection. It is simple and easy to prepare and to modify and relatively safe compared to viral vectors. In recent years, PEI has been utilized in many research areas for gene delivery to stem cells in vitro or targeted gene delivery to cells in the brain. This review reveals that the cytotoxicity and low transfection efficiency of PEI requires to be improved. However brain-targeted modification indicates the promising prospect of PEI for gene therapy in cerebrovascular diseases.
Genetic Therapy ; Genetic Vectors ; Humans ; Nanostructures ; chemistry ; Polyethyleneimine ; chemistry ; Stem Cell Transplantation ; methods ; Transfection ; methods

Carcinoma, Hepatocellular ; therapy ; Genetic Therapy ; methods ; Humans ; Liver Neoplasms ; therapy ; Transfection

We developed a detailed electroporation method to deliver efficiently siRNA into mouse preimplantation embryos. By introducing Cy3 labeled negative control small interfering RNA (siRNA) into mouse preimplantation embryos, we optimized conditions for the electroporation, including the voltage, pulse duration, pulse number, electroporation buffer and an important step to weaken the zona pellucida. Embryonic survival rate, transfection rate and blastocyst development rate were evaluated under the converted fluorescence microscope, by embryos counting and statistical analysis. The best transfection was achieved in opti-MEM under the conditions of 30 V, 1 ms, 3 pulses, and the duration of digestion in tyrode's solution was 10 s. In conclusion, the proposed electroporation approach here is a simple and efficient tool to deliver siRNA for RNA interference (RNAi) into mouse preimplantation embryos.
Animals ; Blastocyst ; metabolism ; Electroporation ; Female ; Male ; Mice ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection ; methods

OBJECTIVETo investigate endostatin gene therapy of rat corneal neovascularization induced by acid cauterization.

METHODSpBlast-hEndostatin and pBlast-Mcs were identified by digestion with Nhe Iand Sal I, by PCR reaction, by sequence, and then by alignment of PCR products with the gene Bank using NCBIBLAST software. They were then purified with QIAGEN Endofree plasmid maxi kit. Rat corneal neovascularization models were made with 75% AgNO(3) and 25% KNO(3) cauterization. The treatment method was subconjunctive injection of the pBlast-hEndostatin with the control of pBlast-Mcs.

RESULTSpBlast-hEndostatin was found to contain the human endostatin gene. The rat corneal neovascularization induced by acid cauterization was significantly suppressed after subconjunctive injection of the pBlast-hEndostatin with inhibition rates of 37%, 40.2%, and 42.8% respectively on the sixth, tenth, and fifteenth day. The inhibition rate for the density of corneal neovascularization was 40%. However, no inhibition effect on the length of the neovascularization and corneal inflammatory cells was observed. Corneal neovascularization areas were positively correlated with edema and corneal opacity.

CONCLUSIONSThe plasmid of pBlast-hEndostatin contained the human endostatin gene. The rat corneal neovascularization induced by acid cauterization can be partially inhibited by subconjunctive injection of the pBlast-hEndostatin mediated by liposomes. Endostatin produced by transfected fibroblast cells directly inhibits corneal neovascularization. This is not caused by inflammatory reaction inhibition.

Animals ; Corneal Neovascularization ; pathology ; therapy ; Endostatins ; genetics ; Genetic Therapy ; methods ; Humans ; Rats ; Rats, Wistar ; Transfection

OBJECTIVETo develop a RP-HPLC method for the determination of quercetin in UGT1A3 cDNA-transfected cells.

METHODSThe lysate of cells transfected with human recombinant uridine 5-diphosphate glucuronosyltransferases UGT1A3 cDNA was co-incubated with quercetin, the reaction was terminated with acetonitrile, and luteolin was used as internal standard. The determination was performed on a C(1) reversed phase column with a mobile phase of methanol-0.1% formic acid (V/V) at a flow rate of 1.0 ml/min. The gradient elution was as follows: 0 - 25 min (30:70-80:20, methanol:0.1% formic acid), > 25-25.5 min (80:20), >25.5-27 min (80:20-30:70), > 27-30 min (30:70). A UV-VIS detector was operated at 368 nm.

RESULTThe standard curve was linear over the concentration range of 5-200 μmol/L (r = 0.9999). The limit of detection was 1.25 μmol/L(S/N ≥ 3), and the limit of quantification was 5 μmol/L (S/N >10, RSD = 6.99%). The method afforded recoveries of 99.1%-103.5%, and precisions for inter- and intra-assay were < 2.5% and < 8%, respectively. In addition, kinetic analysis indicated that the K(m), V(max) and CL(int) (V(max)/K(m)) values for quercetin glucuronide were (62.95 ± 13.16) μ mol/L, (284.50 ± 24.35)nmol*min⁻¹*g⁻¹ and 4.52 ml*min⁻¹*g⁻¹, respectively.

CONCLUSIONThe method established is accurate and simple and suitable for the determination of quercetin in UGT1A3 cDNA-expressed cells.

Cells, Cultured ; Chromatography, High Pressure Liquid ; methods ; Glucuronosyltransferase ; genetics ; Humans ; Quercetin ; analysis ; pharmacokinetics ; Transfection

OBJECTIVETo observe the effect of plasmids in different size and gene transfection protocol on efficiency of introducing gene into human KC.

METHODSFour plasmids in different size, inclu-ding pSUPER-enhanced green fluorescent protein (EGFP), pEGFP-N2, pHSER-green fluorescent protein (GFP) and ploxP-EGFP, were transfected into immortal human KC line (HaCaT) and human embryo kid-ney cell line (293FT) separately following transfection protocols of liposome (LTP), cation polymerizer (CPTP), electroporation combined with nucleus transfection agent (ETP) and lentivirus. 293FT was used as control. GFP expression was observed under inverted fluorescence microscope. The transfection efficiency (TE) was calculated.

RESULTS(1) The four plasmids could be introduced into HaCaT (TE, 1.0%-3.3%) and 293FT (TE, 80.0%-84.7% ) following LTP. (2) The four plasmids could also be introduced into HaCaT (TE, 1.0%-3.7% ) and 293FT (TE, 81.3%-86.7% ) following CPTP. (3) Two shorter plasmids (pSUPER-EGFP and pEGFP-N2) could be introduced into HaCaT by ETP with higher TE than the othr two longer plasmids (pHSER-GFP and ploxP-EGFP), which were 22.3% and 19.0% vs. 4.0% and 3.3%, respectively. (4) pHSER-GFP packaged by lentivirus could be introduced into HaCaT with the TE reaching 97.0%, which surpassed the above three protocols.

CONCLUSIONSIt is difficult to introduce exogenous gene into human KC by LTP or CPTP; TE of lentivirus transfection protocol apparently surpasses

Cell Line ; Genetic Therapy ; methods ; Genetic Vectors ; Humans ; Keratinocytes ; Liposomes ; metabolism ; Plasmids ; Transfection

OBJECTIVETo study total nutrient admixture (TNA) promoting plasmid DNA transfection mediated with liposomes to colorectal cancer cells.

METHODSDispensing varied transfection agents of liposome + DNA plasmid pEGFP-N(1), TNA + liposomes + pEGFP-N(1), TNA + pEGFP-N(1), liposomes merely, and TAN sole. Human colorectal cancer cell LoVo and HR-8348 were treated with the agents respectively. Green fluorescence protein (GFP) gene as a report gene was detected.

RESULTSGFP was not detected in cancer cells treated with agents of merely liposomes and TAN. Transfection rates of GFP in two groups of cancer cells treated with agent of TNA + liposomes + pEGFP-N(1) were 33%, 38% respectively. With liposome + pEGFP-N(1), the rates of transfection in two cells were 22%, 24% respectively. The expression of GFP was 1% in the two groups of tumor cells treated with TNA + pEGFP-N(1). With agent of TNA + liposomes + pEGFP-N(1), a high transfection rate of GFP gene was obtained. And no negative effect was observed to stabilization of TAN solution.

CONCLUSIONTNA may enhance transfection rate of plasmid DNA mediated with liposome, and may be beneficial to the treatment of cancer.

Colorectal Neoplasms ; therapy ; Genetic Therapy ; Humans ; Liposomes ; Nutritional Support ; Plasmids ; Transfection ; methods

OBJECTIVETo investigate the effect of electroporation-mediated gene transfect on the expression of cyclins during mandible distraction in rabbit.

METHODSBilateral mandibular osteotomy was performed in 45 New-Zeland rabbits. After a latency of 3 days, the mandibles were elongated using distractors with a rate of 0.8 mm/day for 7 days. After the completion of distraction, the rabbits were randomly divided into 5 groups. 2 microg (0.1 microg/microl) of pIRES-hVEGF165-hBMP2, recombinant plasmid pIRES-hBMP2, recombinant plasmid pIRES-hVEGF165, pIRES and the same volume of normal saline (NS) was injected into the distraction area in each group, respectively. After injection, electroporation was performed in every group. Three animals in each group were sacrificed at 7, 14, and 28 days after completion of distraction, respectively. The lengthened mandibles were harvested and processed for immunohistochemical examinations. The expression of cyclins A, D1 ,E in positive cells were measured by CMIAS-2001A computerized image analyzer. The data were analyzed with the single factor analysis of variance and q test.

RESULTSCyclins A, D1, E staining was mainly located in inflammatory cells, granulation tissue monocyte, fibroblast, osteoblasts, osteocyte and the connective tissues around the new bone. The expression reached to the peak at 7th day of consolidation, and decreased at 14th day, and weak at 28th day. Image analysis results showed that, at 7th day, the expression absorbance A in group C (0.59 +/- 0.14) was the strongest, compared to group A (0.41 +/- 0.13), B (0.38 +/- 0.14), D (0.34 +/- 0.12) and E (0.31 +/- 0.10), showing a significant difference (P < 0.05, P < 0.01). There was no significance difference between group A and B (P > 0.05), but the difference between group A/B and group D/E (P < 0.05). At 14th and 28th day, there was no significant difference among group A (0.39 +/- 0.11), B (0.34 +/- 0.10) and C (0.33 +/- 0.09) (P > 0.05), but there was significant difference between group A/B/C and group D (0.19 +/- 0.12) or E (0.14 +/- 0.04) (P < 0.05 or P < 0.01).

CONCLUSIONSElectroporation-mediated gene transfection can promote cyclins A, D1, E expression effectively, which may promote cell differentiation and proliferation, stimulate extracellular matrix synthesis and new bone formation in distraction gap.

Animals ; Cyclins ; metabolism ; Electroporation ; Genetic Therapy ; Mandible ; surgery ; Osteogenesis, Distraction ; methods ; Plasmids ; Rabbits ; Transfection