BACKGROUNDIt has been proven that ultrasonic destruction of microbubbles can enhance gene transfection efficiency into the noncardiac cells, but there are few reports about cardiac myocytes. Moreover, the exact mechanisms are not yet clear; whether the characteristic of microbubbles can affect the gene transfection efficiency or not is still controversial. This study was designed to investigate whether the ultrasound destruction of gene-loaded microbubbles could enhance the plasmids carried reporter gene transfection in primary cultured myocardial cell, and evaluate the effects of microbubbles characteristics on the transgene expression in cardiac myocytes.

METHODSThe β-galactosidase plasmids attached to the two types of microbubbles, air-contained sonicated dextrose albumin (ASDA) and perfluoropropane-exposed sonicated dextrose albumin (PESDA) were prepared. The gene transfection into cardiac myocytes was performed in vitro by naked plasmids, ultrasound exposure, ultrasonic destruction of gene-loaded microbubbles and calcium phosphate precipitation, and then the gene expression and cell viability were analyzed.

RESULTSThe ultrasonic destruction of gene-loaded microbubbles enhanced gene expression in cardiac myocytes compared with naked plasmid transfection ((51.95 ± 2.41) U/g or (29.28 ± 3.65) U/g vs. (0.84 ± 0.21) U/g, P < 0.01), and ultrasonic destruction PESDA resulted in more significant gene expression than ASDA ((51.95 ± 2.41) U/g vs. (29.28 ± 3.65) U/g, P < 0.05). Ultrasonic destruction of microbubbles during calcium phosphate precipitation gene transfection enhanced &beta;-galactosidase activity nearly 8-fold compared with calcium phosphate precipitation gene transfection alone ((111.35 ± 11.21) U/g protein vs. (14.13 ± 2.58) U/g protein, P < 0.01). Even 6 hours after calcium phosphate precipitation gene transfection, ultrasound-mediated microbubbles destruction resulted in more intense gene expression ((35.63 ± 7.65) U/g vs. (14.13 ± 2.58) U/g, P < 0.05).

CONCLUSIONSUltrasonic destruction of microbubbles might be a promising method for the delivery of non-viral DNA into cardiac myocytes, and the gene tranfection is related to the characteristics of microbubbles.

Albumins ; Animals ; Cell Survival ; genetics ; physiology ; Cells, Cultured ; Microbubbles ; Myocytes, Cardiac ; cytology ; metabolism ; Rats ; Rats, Wistar ; Transfection ; methods ; Ultrasonics ; methods ; beta-Galactosidase ; genetics ; metabolism

OBJECTIVETo study the content of phytoestrogen in dissimilarity herbs.

METHODThe activity of phytoestrogen in heat-clearing drugs, drugs for relieving exterior syndrome, diuretic, anastaltics, tonics and astringents were detected based on the recombinant yeast cell (W303-1A/hER-ERE-Lac Z). The estrogenic activity in traditional Chinese materia medica were assayed quantitatively by determining the expression of beta-galactosidase.

RESULTThe phytoestrogen concentration (6.35 x 10(-3) nmol x g(-1) E2 equivalent) in heat-clearing drugs was the highest while that in anastaltic and tonic drugs was the lowest, which was less than the detected limit.

CONCLUSIONCompared with the other traditional Chinese materia medica, the content of phytoestrogen, which can bind to estrogen receptor, in giant knotweed rhizome, forsythia suspense, ash bark, baical skullcap root and ophiopogonis tuber were higher.

Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Phytoestrogens ; analysis ; metabolism ; Plants, Medicinal ; chemistry ; Receptors, Estrogen ; metabolism ; Recombination, Genetic ; Saccharomyces cerevisiae ; chemistry ; cytology ; drug effects ; genetics ; beta-Galactosidase ; analysis

OBJECTIVETo explore a way of the gene therapy for acute spinal cord injury (ASCI) by vivo transfection of exogenous gene into spinal cord tissue.

METHODSTwenty-four rats of SD were divided into experiment group and control group (each group had 12 rats). After anaesthesia by abdominal cavity, lamina of thoracic vertebra of all rats were cut-open in prone position. Complex of plasmid and report gene-Lac Z, and plasmid without report gene-Lac Z were respectively injected into cavum subdural of SD rats of experiment group and control group by cation liposome (DOTAP) encapsulation. The rats were killed at the 2nd week after operation, spinal cord tissue of injected segments were detected by reverse transcription-polymerase chain raction (RT-PCR) and immunohistochemistry.

RESULTSIn experiment group, positive staining of beta-galactosidase can be clearly observed in neuron and glia cell of rat's spinal cord by immunohistochemistry detection. Lac Z mRNA in same area was also detected by RT-PCR. But, in control group, no above-mentioned positive results were found.

CONCLUSIONEffective transfection of exogenous gene in vivo into spinal cord is a new hot spot for treatment of SCI. Thus certain nerve growth factor imput partly area of spinal cord injury can promote central nerve regrowth and avoid early secondary injury.

Acute Disease ; Animals ; Genetic Therapy ; Lac Operon ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Spinal Cord ; metabolism ; Spinal Cord Injuries ; therapy ; Transfection ; beta-Galactosidase ; analysis

For metabolic engineering of cyanobacteria, there is an urgent need to construct a group of efficient heterologous gene expression platforms and to evaluate their expression efficiencies. Here we constructed three integrative vectors, the pKW1188-derived pFQ9F, pFQ9R and pFQ20, for integration of heterologous genes into the genome of the model cyanobacteria strain Synechocystis sp. strain PCC6803. The pFQ16, an RSF1010-derived broad host range shuttle vector, was constructed for conjugative transfer of genes to various cyanobacteria strains. All the four platforms constructed here applied the rbc (encodes Ribulose-1, 5-bisphosphate carboxylase/oxygenase) and the rbc terminator to promote and terminate the gene transcription. Besides, a "Shine-Dalgarno -AUG" fusion translation strategy was used to keep the high protein translation efficiency. Using lacZ as a reporter gene, the expression efficiency of pFQ20 was evaluated and showed a strong beta-galactosidase expression (109 Miller). Furthermore, the platform pFQ20 was used to express the E. coli tesA' gene and showed significant protein bands through the Western Blot test. The expression platforms constructed in this study offer useful molecular tools for metabolic engineering of cyanobacteria in the future.
Genetic Vectors ; genetics ; Industrial Microbiology ; methods ; Metabolic Engineering ; methods ; Palmitoyl-CoA Hydrolase ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Synechocystis ; genetics ; metabolism ; beta-Galactosidase ; biosynthesis ; genetics

To improve the stability and gene-carried capability of gene-attached microbubbles, the method for manufacture of albumin microbubbles was modified and new gene-loaded microbubbles were synthesized by incorporated gene-PEI complex into the shell of microbubbles. Agarose gel electrophoresis and bacteria transformation showed that PEI had the ability to provide the protection of plasmid DNA from ultrasonic degradation. The new gene-loaded microbubbles exhibited excellent acoustical and hemorheological properties. Moreover, they could carry more plasmid DNA than gene-attached microbubbles. beta-galactosidase plasmid transfection into cardiac myocytes was performed by using ultrasound targeted destruction of new gene-loaded microbubbles or gene-attached microbubbles. Gene expression in cardiac myocytes was detected by beta-galactosidase in situ staining and quantitive assay. It was shown that beta-galactosidase activity in cardiac myocytes was enhanced 107-fold by ultrasonic destruction of gene-loaded microbubbles compared with naked plasmid transfection and new gene-loaded microbubbles resulted in 6.85-fold increase in beta-galactosidase activity compared with optimal transfection mediated by gene-attached microbubbles. These results suggested that ultrasonic destruction of the gene-loaded microbubbles can enhance the cardiac myocytes exogenous gene transfer efficiency significantly and new gene-loaded microbubbles is an efficient and safe gene delivery vehicle.
Animals ; Cells, Cultured ; Genes, Reporter ; genetics ; Genetic Vectors ; Imines ; Microbubbles ; Myocytes, Cardiac ; metabolism ; Plasmids ; genetics ; Polyethylenes ; Rats ; Rats, Wistar ; Sonication ; Transfection ; methods ; beta-Galactosidase ; biosynthesis ; genetics

Enhanced extracellular matrix (ECM) accumulation is an important finding in human restenotic arterial neointima after angioplasty. Transforming growth factor b1(TGF-beta1) is known to regulate the synthesis and turnover of a variety of ECM components, and may play an important role in restenosis. Recombinant adenoviral vector expressing an ectodomain of the TGF-beta type II receptor fused to the human immunoglobulin Fc portion (AdT beta-ExR) inhibits the action of TGF-beta probably either by adsorbing TGF-beta or by acting as a dominant negative receptor. We carried out a catheter-based local adenovirus mediated gene delivery using an Infiltrator in porcine coronary arteries to know the pattern of gene expression, efficacy and procedural complications. Twenty four coronary arteries in 13 pigs were used for intravascular gene delivery by intramural injection with either AdT beta-ExR or adenovirus expressing b-galactosidase (AdCALacZ). Direct immunofluorescent staining and reverse transcription polymerase chain reaction (RTPCR) were used for detection of type II TGF-beta receptor and its mRNA respectively. X-Gal histochemistry was performed to identify b-galactosidase. Both soluble TGF-beta receptor and b-galactosidase were expressed locally in the media and adventita at injected arterial segments without any significant dissemination to remote area. Intravascular gene transfection performed with various titer of each adenoviral vector showed that AdT beta-ExR of 5x10(8) pfu and AdCALacZ of 2.5 x 10(8) pfu were the minimum titer for the expression of each transgene. Infiltration of CD3 positive T cells was detected by immunohistochemical staining in the area of each transgene expression, and tends to decrease over time after gene delivery. Pathological study of 24 treated arteries showed complications such as disruption of external elastic lamina with hemorrhage (n = 4), minimal disruption of internal elastic lamina and endothelial layer, and medial thickening. In conclusion, catheter-based local intravascular gene delivery of adenoviral vector is feasible and effective in a selected artery, but must be undertaken with caution due to possible lethal complications. Local delivery of soluble TGF-beta type II receptor in this way may provide an effective intravascular gene therapy to inhibit TGF-beta signal pathway without any significant systemic side effect.
Adenoviridae/*genetics ; Animals ; *Catheters, Indwelling ; Coronary Vessels/*metabolism/pathology ; Female ; Gene Expression ; Gene Therapy/*adverse effects/*methods/mortality ; *Gene Transfer Techniques ; Genetic Vectors/*administration & dosage ; Inflammation/etiology ; Receptors, Transforming Growth Factor beta/analysis/*metabolism ; Swine ; Transgenes ; beta-Galactosidase/genetics/metabolism

Enhanced extracellular matrix (ECM) accumulation is an important finding in human restenotic arterial neointima after angioplasty. Transforming growth factor b1(TGF-beta1) is known to regulate the synthesis and turnover of a variety of ECM components, and may play an important role in restenosis. Recombinant adenoviral vector expressing an ectodomain of the TGF-beta type II receptor fused to the human immunoglobulin Fc portion (AdT beta-ExR) inhibits the action of TGF-beta probably either by adsorbing TGF-beta or by acting as a dominant negative receptor. We carried out a catheter-based local adenovirus mediated gene delivery using an Infiltrator in porcine coronary arteries to know the pattern of gene expression, efficacy and procedural complications. Twenty four coronary arteries in 13 pigs were used for intravascular gene delivery by intramural injection with either AdT beta-ExR or adenovirus expressing b-galactosidase (AdCALacZ). Direct immunofluorescent staining and reverse transcription polymerase chain reaction (RTPCR) were used for detection of type II TGF-beta receptor and its mRNA respectively. X-Gal histochemistry was performed to identify b-galactosidase. Both soluble TGF-beta receptor and b-galactosidase were expressed locally in the media and adventita at injected arterial segments without any significant dissemination to remote area. Intravascular gene transfection performed with various titer of each adenoviral vector showed that AdT beta-ExR of 5x10(8) pfu and AdCALacZ of 2.5 x 10(8) pfu were the minimum titer for the expression of each transgene. Infiltration of CD3 positive T cells was detected by immunohistochemical staining in the area of each transgene expression, and tends to decrease over time after gene delivery. Pathological study of 24 treated arteries showed complications such as disruption of external elastic lamina with hemorrhage (n = 4), minimal disruption of internal elastic lamina and endothelial layer, and medial thickening. In conclusion, catheter-based local intravascular gene delivery of adenoviral vector is feasible and effective in a selected artery, but must be undertaken with caution due to possible lethal complications. Local delivery of soluble TGF-beta type II receptor in this way may provide an effective intravascular gene therapy to inhibit TGF-beta signal pathway without any significant systemic side effect.
Adenoviridae/*genetics ; Animals ; *Catheters, Indwelling ; Coronary Vessels/*metabolism/pathology ; Female ; Gene Expression ; Gene Therapy/*adverse effects/*methods/mortality ; *Gene Transfer Techniques ; Genetic Vectors/*administration & dosage ; Inflammation/etiology ; Receptors, Transforming Growth Factor beta/analysis/*metabolism ; Swine ; Transgenes ; beta-Galactosidase/genetics/metabolism

The present study was aimed to investigate the effect of pretreatment with hydrogen sulfide (H2S) on human umbilical vein endothelial cells (HUVECs) senescence and the underlying mechanism. Cultured HUVECs at twelfth and fourth passages were taken as old and young groups, respectively. Sodium hydrosulfide (NaHS, donor of H2S) group was treated with NaHS from fourth to twelfth passage. The cell senescence was determined by senescence-associated &beta;-galactosidase (SA &beta;-gal) staining. DAPI fluorescent dye was used to detect cellular apoptosis. Western blot was used to analyze the expression levels of xanthine oxidase (XOD), manganese-superoxide dismutase (Mn-SOD) and the subunits p67(phox) of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the HUVECs. Colorimetric method was used to detect SOD activity and cellular hydrogen peroxide (H2O2) level. The results showed that, compared with young group, the old group exhibited higher SA &beta;-gal positive rate and cellular apoptosis, while NaHS pretreatment decreased SA &beta;-gal positive rate and cellular apoptosis. Compared with the young group, the old group showed increased expression levels of XOD and p67(phox), as well as lower Mn-SOD expression level. With the pretreatment of NaHS, the up-regulations of XOD and p67(phox) levels and down-regulation of Mn-SOD level were inhibited. Compared with the young group, the old group showed lower SOD activity and higher H2O2 level, whereas NaHS pretreatment reversed the changes of SOD activity and H2O2 level. These results suggest that H2S delays senescence of HUVECs through lessening oxidative stress.
Apoptosis ; Cellular Senescence ; drug effects ; Down-Regulation ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; Humans ; Hydrogen Peroxide ; metabolism ; Hydrogen Sulfide ; pharmacology ; Oxidative Stress ; Phosphoproteins ; metabolism ; Superoxide Dismutase ; metabolism ; Xanthine Oxidase ; metabolism ; beta-Galactosidase ; metabolism

OBJECTIVETo investigate the synergetic transactivating functions of HCV core and truncated HBV middle surface proteins.

METHODSTwo recombinant expression plasmids harboring HCV core and C-terminally truncated HBV middle surface protein gene were constructed, respectively. The plasmids were transfected into HepG2 cells and cotransfected HepG2 cells with reporter plasmid pSV-lacZ by lipofectamine plus reagents. The transient expressed viral proteins were identified at the transcription and translation levels. The activity of beta-galactosidase was detected, which reflected the transactivating function of the proteins.

RESULTSThe protein expression of plasmids was detected in soluble cell extracts of transiently transfected HepG2 cells. HCV core protein activated the beta-galactosidase expression at a value of 4.6 times higher than the control, while C-terminally truncated HBV middle surface protein activated at a value of 3.2 times. It reached 8.4 times transfected with the plasmids simultaneously. The transactivating effect was dose dependent.

CONCLUSIONSIt is suggested that the two kinds of virus proteins have transactivating effect on SV40 early promoter/enhancer, and they act synergistically. These contribute to explain the mechanisms of liver injury or tumorigenesis induced by HCV or/and HBV infection.

Hep G2 Cells ; Hepatitis B ; genetics ; metabolism ; Hepatitis C ; genetics ; metabolism ; Humans ; Membrane Proteins ; genetics ; metabolism ; Plasmids ; Promoter Regions, Genetic ; Transcriptional Activation ; Transfection ; Viral Core Proteins ; genetics ; metabolism ; beta-Galactosidase

To investigate the role and mechanism of Rac1 protein in the process of the human umbilical vein endothelial cell (HUVEC) senescence, we used hypoxia as a model for modulating HUVECs entering replicative senescence in vitro. Premature senescence of HUVECs was evidenced by detecting the SA-beta-Gal activity and PAI-1 expression. Meanwhile, cell cycle distribution and cell proliferation rate were investigated by flow cytometry assay and BrdU staining. The results indicated that the HUVECs became enlarged and flattened, both SA-beta-Gal activity and PAI-1 expression increased obviously, while cell proliferation was inhibited and G(1) phase cell cycle arresting occurred when HUVECs were treated with continued hypoxia for 96 h. Accompanied with these changes, the expression of activated Rac1 increased obviously in cells after hypoxia. All these observations suggested that endothelial senescence could be induced by continued hypoxia and it might correlate with the activity of Rac1. To further define the relationship between Rac1 and HUVEC senescence, HUVECs were transiently infected with the constitutively active form of Rac1 (V12Rac1) or dominant negative form of Rac1 (N17Rac1) using retrovirus vector pLNCX-V12Rac1 or pLNCX-N17Rac1. We observed the changes of these three kinds of HUVECs (HUVECs, N17Rac1-HUVECs, V12Rac1-HUVECs) after hypoxia for 48 h and 96 h, the expression and localization of serum response factor (SRF), which is one of the downstream signal molecules of Rac1, were also investigated. The results obtained indicated that after continued hypoxia for 48 h, HUVECs infected by V12Rac1 showed obvious senescence accompanied with SA-beta-Gal activation, PAI-1 expression increase, G(1) phase arrest and cell proliferation inhibition which were similar to HUVECs after continued 96-hour hypoxia treatment, while the senescence of HUVECs infected by N17Rac1 was significantly inhibited even if the cells were exposed to hypoxia for more than 96 h. All the results identified that the activation of Rac1 might accelerate HUVEC senescence induced by hypoxia and that inactivation of Rac1 could partly block the cell senescence. To further investigate the mechanism of HUVEC senescence induced by Rac1, we detected the expression of total SRF (tSRF) and nuclear SRF (nSRF) in these three kinds of HUVECs by immunofluorescent analysis and Western blot assay after hypoxia. The results showed that the expression of nSRF decreased obviously and the nuclear translocation of SRF was inhibited in HUVECs infected by V12Rac1 compared with those in the normal HUVECs. In contrast, the expression of nSRF increased obviously in the HUVECs infected by N17Rac1. These results suggest that activation of Rac1 accelerates endothelial cell senescence and inhibition of Rac1 activity prevents HUVECs from entering senescence induced by hypoxia, while the nuclear translocation of SRF regulated by Rac1 might play an important role in the process of senescence.
Cell Hypoxia ; Cells, Cultured ; Cellular Senescence ; physiology ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; Plasminogen Activator Inhibitor 1 ; genetics ; metabolism ; Serum Response Factor ; genetics ; metabolism ; beta-Galactosidase ; metabolism ; rac1 GTP-Binding Protein ; metabolism