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.

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.

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.

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

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.

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.

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.

Differentiation of Rabbit Bone Marrow Mesenchymal Stem Cells to Myogenic Cells and Expression of VEGF After Gene Transfection

Objectives To induce the differentiation of rabbit bone marrow mesenchymal stem cells （MSCs） to myogenic cells in vitro, and to investigate the expression of vascular endothelial growth factor （VEGF） gene in MSCs transfected by AdTrackCMV-VEGF165. Methods MSCs were isolated and purified from rabbit bone marrow by percoll （11）73 g/ml） and then cultured in low glucose DMEM with 10% FBS. AdTrackCMV-VEGF165 eukaryotic expression vector was constructed and transfected into the MSCs. After being incubated with 5-azacytidine （5- Aza）, the expression of troponin I in MSCs was assayed by immunohistochemistry and the expression of VEGF gene was identified by northern blot and western blot. The concentration of VEGF in the supernatant was measured by ELASA. Results MSCs were isolated and cultured successfully from rabbit bone marrow. The positive cTnI stain of some MSCs after the induction of 5-aza indicated that the cells were differentiated to myogenic cells. Northern blot and western blot showed that the expression of VEGF 165 mRNA was much higher in the hVEGF165 gene transfected cells than that of the control. The concentration of VEGF in the supernatant got to the peak 3-5 days after hVEGF165 gene transfection （1011- 1027 pg/ml） and decreased gradually thereafter, but still higher than that of control group or pAdTrackCMV group （349 pg/mLvs 116 pg/mL or 125pg/ml respectively, MSCs could be induced P〈 0.01）. Conclusions to differentiate to myogenic cells by 5-aza in vitro and could express VEGF by VEGF gene transfection.

Acceleration of apoptosis by transfection of bak gene in multi-drug resistant (MDR) bladder cancer cellsI

Objective To observe the effects of bak gene on killing MDR bladder cancer cells and to study its mechanisms. Methods Bak gene was transfected into MDR bladder cancer cells by liposome. The mRNA of bak and bcl-2 were detected by in situ hybridization. The protein of bak and bcl-2 were detected by SABC immunohistochemistry. The growth rate of human bladder cancer cells was studied by constructing the growth curve, cell apoptosis being observed by flow cytometry, and the outline of cells observed by fluorescence stain. Results The expression of bak mRNA was positive in EJ/bak cells (64% ,P【0.05).Bak protein expression of EJ/bak cells was positive (60 % ) and bcl-2 protein expression was de creased (P【0.05). The growth of MDR bladder cancer cells was significantly inhibited by 32% after bak gene was transfected (P 【 0. 05 ). Apoptosis cells increased significantly. The apoptosis rate was 35 %. Apoptotic bodies can be found in these cells on fluorescence stain. Conclusion Bak gene could inhibit the growth

Cardiac autonomic nerve fiber regeneration in chronic heart failure Do Akt gene-transduced mesenchymal stem cells promote repair?

BACKGROUND： Transplantation of Akt-over-expressing mesenchymal stem ceils （Akt-MSCs） has been shown to repair infarcted myocardium and improve cardiac function. However, little is known about the therapeutic effects of Akt-MSCs on cardiac autonomic neuropathy in chronic heart failure （CHF）. OBJECTIVE： The present study used adriamycin-induced CHF rat models to observe the effect of Akt-MSCs on cardiac autonomic nervous regeneration and the factors mediating this effect. DESIGN, TIME AND SETTING： A randomized, controlled animal experiment was performed at the Central Laboratory of Basic Medical College, China Medical University, between September 2008 and April 2009. MATERIALS： Rabbit anti-choline acetyltransferase （CHAT）, growth associated protein-43 （GAP-43） synaptophysin （SYN） polyclonal antibodies and the secondary antibody （goat anti-rabbit IgG） were purchased from Boster, China. Cat-A-Kit assay system was provided by Amersham, USA. METHODS： （1） Adult rat MSCs were isolated and cultured for the preparation of Akt-MSCs. （2） Forty male Wistar rats were intramyocardially administered adriamycin at 2 mg/kg over 3 days for a total of five times and once a week for additional five times thereafter to establish CHF models. At 2 weeks after final adriamycin treatment, 34 successful CHF rat models were randomized to three groups： Akt-MSCs （n = 11）, simple MSCs （s-MSCs, n =11）, and control （n = 12）. Each group was intravenously administered Akt-MSCs （2x106 cells in 100 IJL PBS）, s-MSCs （2×10^6 cells in 100 μL PBS） or equal volume of phosphate buffered saline, once a day for a total of three times. MAIN OUTCOME MEASURES： At 4 weeks after final adriamycin treatment, myocardial norepinephrine （NE） content was detected using a Cat-A-Kit assay system. Myocardial CHAT, SYN and GAP-43 were performed by immunohistochemistry and Western blot analysis. Prior to, 2 and 4 weeks after adriamycin treatment, echocardiographic examination was performed and left ventricular ejection fraction （LVEF） was determined. RESULTS： Myocardial NE content, as well as SYN-positive and GAP-43-positive nerve fiber density and expression, and LVEF, was the greatest in the Akt-MSCs group, followed by the s-MSCs group, and lastly the control group （P 〈 0.05 or P 〈 0.01）. ChAT expression was similar between Akt-MSCs and s-MSCs groups, but it was higher compared with the control group （P 〈 0.05）. NE contents were negatively correlated to LVEF （r = -0.64, P = 0.015）. CONCLUSION： Transplantation of MSCs, in particular Akt-MSCs, promotes cardiac nervous regeneration in failing heart, which might be mediated by GAP-43.

Surface Modification of Biomimetic PLGA-(ASP-PEG) Matrix with RGD-Containing Peptide:a New Non-Viral Vector for Gene Transfer and Tissue Engineering

RGD-containing peptide （ K16-GRGDSPC） , characterized as non-viral gene vectors, was fabricated to modify the surface of PLGA-[ASP- PEG] matrix, which offered the foundation for gene transfer with porous matrix of gene activated later. Peptide was synthesized and matrix was executed into chips A, B and chip C. Chip C was regarded as control. Chips A and B were reacted with cross-linker. Then chip A was reacted with peptide. MS and HPLC were ased to detect the .14W and purity of peptide. Sulphur, existing on the surface of biomaterials, was detected by XPS. The purity of un-reacted peptide in residual solution was detected by a spectrophotometer. HPLC shows that the peptide purity was 94%- 95% , and MS shows that the MW was 2 741. 3307. XPS reveals that the binding energy of sulphur was 164 eV and the ratio of carbon to sulphur （C/S） was 99. 746 ：0. 1014 in reacted chip A. The binding energy of sulphur in reacted chip B was 164 eV and 162 eV, C/ S was 99.574：0.4255, aM there was no sulphur in chip C. Peptide was manufactured and linked to the surface of biomimetic and 3-D matrix, which offered the possibilities for gene transfer and tissue engineering with this new kind of non-viral gene vector.

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.

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.

Use of adenovirus vector expressing the mouse full estrogen receptor alpha gene to infect mouse primary neurons

Estrogen plays important regulatory and protective roles in the central nervous system through estrogen receptor a mediation. Previous studies applied eukaryotic expression and lentiviral vectors carrying estrogen receptor a to cladfy the underlying mechanisms. In the present study, an adenovirus vector expressing the mouse full estrogen receptor a gene was constructed to identify biological characteristics of estrogen receptor a recombinant adenovirus infecting nerve cells. Primary cultured mouse nerve cells were first infected with estrogen receptor a recombinant adenovirus at various multiplicities of infection, followed by 100 multiplicity of infection. Results showed overexpression of estrogen receptor α mRNA and protein in the infected nerve cells. Estrogen receptor a recombinant adenovirus at 100 multiplicity of infection successfully infected neurons and upregulated estrogen receptor a mRNA and protein expression.

The study of human PDGF-B gene transferred to cat corneal endothelial cells

AIM: To demonstrate that human platelet-derived growth factor-B (PDGF-B) cDNA could be Expressed in primary cultured cat corneal endothelia cells by using gene transfer techniques; to explore a useful tool for the further studies of the molecular mechanisms of corneal endothelium failure and provide a potential effective genetic therapy for the blind patients. ' METHODS: Human PDGF-B cDNA was isolated from human placent by RT-PCR and inserted into pcDNA(4) vector to construct recombinant eukaryotic expression plasmid pcDNA(4)-PDGF-B. The full length was confirmed by the DNA sequencing analysis. By tearing endothelium technique we obtained pure single layer of cat corneal endothelial cells. The pcDNA(4)-PDGF-B eukaryotic Expression vector was transferred into cat corneal endothelial cells by Effectene (TM) lipofectine. The transfection efficiency of Effectene (TM) lipofectine in pcDNA(4)-B was detected with pcDNA(4)-GFP. 5 days later, RT-PCR was used to check the PDGF-B expression. Cell viability was tested by modified tertrozalium salt (MU) method. Cell morphology was observed under inverted phase contrast microscope. RESULTS: The human PDGF-B cDNA was isolated successfully from healthy parturien placent tissue and the sequence was confirmed by computer automatic sequence and PCR analysis. Pure single layer cat corneal endothelial cells were successfully cultured by tearing endothelium technique. Effectene (TM) lipofectine transfection technique could be effectively used to transfer pcDNA(4)-PDGF-B into cat corneal endothelial cells in vitro, the transfection efficiency was 30%. RT-PCR result showed that human PDGF-B gene was highly expressed in transfected cat corneal endothelial cells. The expressed PDGF-BB protein promoted the viability of cat corneal endothelial cells. CONCLUSION: Human platelet-derived growth factor-B (PDGF-B) cDNA could be highly Expressed in cultured cat corneal endothelial cells by gene transfection techniques. Expressed PDGF-BB protein significantly promoted the viability of cat corneal endothelial cells, thus provided a potential effective method for corneal endothelium blindness genetic therapy.

Construction of eukaryotic expression vector of human S100A13 gene and its effect on proliferation of human thyroid cancer cell line TT

Objective To investigate the effect of exogenous S100A13 gene overexpression on the proliferation of human thyroid cancer cell line TT.Methods The recombinant ORF of S100A13 tagged with six histidines at the 5' end was subcloned into the pcDNA3.2/V5/GW/D-TOPO vector and sequenced.The eukaryotic expression plasmid pcDNA3.2/V5 /GW/D-S100A13 and empty vector pcDNA3.2/V5/GW/D were transfected into TT cells.The positive clones were selected by G418.The expressions of S100A13 mRNA and protein were detected by real time reverse transcription-polymerase chain reaction(RT-PCR) and Western blot.The effect of S100A13 on cell proliferation and cell cycle was evaluated by cell growth curve,MTT colorimetric assay and flow cytometry.Results S100A13 gene tagged with six histidines at the 5 ' end was confirmed to be inserted into the pcDNA3.2/V5/GW/D vector correctly.TT-S100A13-V5 cells,which over-expressed S100A13,were constructed successfully.TT-S100A13-V5 cells grew much faster than TT-V5 and TT cells(P <0.001).The proportions of both S and G2/M phase cells were significantly higher in TT-S100A13-V5 cells than those in TT-V5 and TT cells(P <0.001).Conclusion The eukaryotic expression vector containing human S100A13 gene has been successfully constructed,which highly expresses S100A13 in TT cells.Exogenous S100A13 gene overexpression accelerates TT cell proliferation and drives the cell cycle progression of TT cells from G0/G1 phase to S and G2/M phases.

Learning and memory changes in rats following exogenous human hepatocyte growth factor gene injection into cerebral ischemic penumbra

Human hepatocyte growth factor can be used to treat cerebral infarction, administered by lateral ventricular, cerebellomedullary cistern or subarachnoid injections. However, the target gene ex-pression product is scarcely found in the ischemic penumbra, but extensively distributes in other regions, increasing the risks of gene therapy. The present study directly transfected hepatocyte growth factor gene into the ischemic penumbra of rats with transient middle cerebral artery occlusion. Immunohistochemical analysis revealed that infarct volume was significantly decreased, hepatocyte growth factor protein expression level and vessel quantity in the ischemic penumbra were significantly increased, and learning and memory were significantly improved.

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.