EGFP Group Research Articles

An IRF2BP member (CgIRF2BP) involved in negative regulation of CgIFNLP expression in oyster Crassostrea gigas

The IRF2BP family of transcription regulators act as corepressor molecules by inhibiting both enhancer-activated and basal transcription involving in many biological contexts. In the present study, an IRF2BP homologue (CgIRF2BP) was identified from oyster C. gigas. Its open reading frame is of 1809 bp encoding a polypeptide of 602 amino acids, which contains an IRF-2BP1_2 domain and a RING domain. The mRNA transcripts of CgIRF2BP were detected in all tested tissues with highest level in haemocytes (28.99-fold of that in mantle, p < 0.05). After poly (I:C) stimulation, the expression level of CgIRF2BP was significantly down-regulated at 3 h (0.50-fold of that in control group, p < 0.001) and gradually increased from 6 h to 48 h (2.69-fold of that in control group, p < 0.01). The recombinant protein of CgIRF2BP (rCgIRF2BP) showed high affinity to both rCgIRF1 and rCgIRF8 with Kd value of 1.02 × 10−7 and 2.09 × 10−7, respectively. In CgIRF2BP-RNAi oysters, the mRNA expression of CgIFNLP, CgMx1, CgViperin and CgIFI44L were significantly increased after poly (I:C) stimulation, which were 2.88 (p < 0.01), 1.83 (p < 0.05), 2.47 (p < 0.05), and 1.99-fold (p < 0.01) of that in EGFP group, respectively. These findings suggested that CgIRF2BP negatively regulated CgIFNLP expression by binding with CgIRF1 and CgIRF8.

The involvement of an interferon-induced protein 44-like (CgIFI44L) in the antiviral immune response of Crassostrea gigas

Interferon-stimulated genes (ISGs) encoding proteins are the essential executors of interferon (IFN) mediated antiviral defense. In the present study, an ISG member, interferon-induced protein 44-like (IFI44L) gene (designed as CgIFI44L-1) was identified from the Pacific oyster Crassostrea gigas. The ORF of CgIFI44L-1 cDNA was of 1437 bp encoding a polypeptide of 479 amino acids with a TLDc domain and an MMR_HSR1 domain. The mRNA transcripts of CgIFI44L-1 were detected in all the tested tissues with highest level in haemocytes, which was 15.78-fold of that in gonad (p < 0.001). Among the haemocytes, the CgIFI44L-1 protein was detected to be highly expressed in granulocytes with dominant distribution in cytoplasm. The mRNA expression level of CgIFI44L-1 in haemocytes was significantly induced by poly (I:C) stimulation, and the expression level peaked at 24 h, which was 24.24-fold (p < 0.0001) of that in control group. After the treatment with the recombinant protein of an oyster IFN-like protein (rCgIFNLP), the mRNA expression level of CgIFI44L-1 was significantly enhanced at 6 h, 12 h and 24 h, which was 2.67-fold (p < 0.001), 5.44-fold (p < 0.001) and 5.16-fold (p < 0.001) of that in control group, respectively. When the expressions of CgSTAT and CgIFNLP were knocked down by RNA interference (RNAi), the mRNA transcripts of CgIFI44L-1 were significantly down-regulated after poly (I:C) stimulation, which was 0.09-fold (p < 0.001) and 0.06-fold (p < 0.001) of those in EGFP group, respectively. These results suggested that CgIFI44L-1 was a conserved ISG in oyster, which was regulated by CgIFNLP and CgSTAT, and involved in the oyster antiviral immune response.

The LOX-1 receptor ectopically expressed in the liver alleviates atherosclerosis by clearing Ox-LDL from the circulation

ObjectiveOxidized Low-Density-Lipoprotein (Ox-LDL) is the core factor in the development of atherosclerosis. However, there are few therapies aimed at eliminating Ox-LDL. Here in this study, we investigate whether the ectopically expression of the lectin-like oxidized low density lipoprotein receptor (LOX-1) in the liver could lead to the elimination of circulating Ox-LDL and prevent the deposition in the vascular wall, thereby alleviating the progression of atherosclerosis.MethodsApolipoprotein E-deficient (ApoE−/−) mice were randomly divided into three groups, the control group, the AAV8-TBG-eGFP group (eGFP group) and AAV8-TBG-LOX-1 group (LOX-1 group). In the LOX-1 group, mice received an injection of virus dilution AAV8-TBG-LOX-1 (1.16 × 1011 virus genome (v.g)/animal/100 μl). The mice in the control group and eGFP group received the same amount of sterile saline and AAV8-TBG-eGFP virus dilution injections. The expression of LOX-1 in the liver was detected by immunofluorescent, western blot and immunohistochemistry. The safety of the virus was assessed by hematoxylin–eosin (H&E) staining, blood biochemical analyses and immunofluorescent. The function of LOX-1 in the liver was detected by the co-localization of LOX-1 and Dil-labeled Ox-LDL (Dil-Ox-LDL) under laser scanning confocal microscope. The extent of Ox-LDL in plasma was detected by ELISA. Changes in blood lipids were assessed through blood biochemical analysis. The progression of atherosclerotic lesions was detected by Oil red O staining. And the expression of Vascular Cell Adhesion Molecule-1 (VCAM-1) in endothelial cells and the extent and migration of macrophages in atherosclerotic plaque were detected by immunofluorescence staining. The protein expression in liver was assessed by qRT-PCR and western blot.ResultsThe expression of LOX-1 was stable in liver within 4 weeks. Ectopically expressed LOX-1 in the liver phagocytosed and degraded Ox-LDL and reduced Ox-LDL from circulation but did not have a significant effect on blood lipid levels. After the expression of LOX-1 in liver, Ox-LDL can be cleared by the hepatocytes, thereby reducing VCAM-1 expression in vascular endothelium and the migration of macrophages in plaques, and eventually alleviating the progression of atherosclerosis. Functional expression of LOX-1 in hepatocytes may facilitate the metabolic clearance of Ox-LDL by upregulating the expression of ATP-binding cassette G5 and G8 (ABCG5/G8), which is the primary neutral sterol transporter in hepatobiliary and transintestinal cholesterol excretion.ConclusionEctopic liver-specific expression of LOX-1 receptor alleviates the progression of atherosclerosis by clearing Ox-LDL from circulation.

A novel CgIFNLP receptor involved in regulating ISG expression in oyster Crassostrea gigas

Interferons (IFNs) are the key coordinators of antiviral immunity by binding to their receptors to orchestrate a complex transcriptional network in vertebrates. Recently, the existence of molluscan IFN-like system has been certified by the identification of important components in IFN system, such as IFN-like protein (CgIFNLP) from oyster Crassostrea gigas. In the present study, a novel CgIFNLP receptor (designed CgIFNLPR-1) was identified from C. gigas. The open reading frame (ORF) of CgIFNLPR-1 cDNA was of 1962 bp encoding a peptide of 653 amino acid residues with five fibronectin type III (FNIII) domains and one transmembrane helix region. The mRNA transcripts of CgIFNLPR-1 were constitutively distributed in all the tested tissues, with the highest level in gonad. After Poly (I:C) stimulation, the mRNA expression of CgIFNLPR-1 in haemocytes was significantly up-regulated to the highest level at 48 h (4.54-fold of that in control group, p < 0.05). CgIFNLPR-1 protein was mainly distributed in the cytoplasm and membrane of oyster haemocytes. CgIFNLP and CgIFNLPR-1 were able to interact with each other in vitro. After the CgIFNLPR-1 was knocked down by RNAi, the mRNA expression of IFN-stimulated genes (ISGs), including CgMx, CgViperin and CgIFNIP-44, were significantly inhibited after Poly (I:C) stimulation, which was 0.17, 0.31 and 0.53-fold of that in EGFP group, respectively (p < 0.01). These findings suggested that CgIFNLPR-1 was a novel CgIFNLP receptor in the oyster to recognize CgIFNLP and regulate the expressions of CgISGs.

Oral immunization with recombinant Lactobacillus casei displayed AHA1-CK6 and VP2 induces protection against infectious pancreatic necrosis in rainbow trout (Oncorhynchus mykiss)

Infectious pancreatic necrosis virus (IPNV) primarily infects larvae and young salmonid with serious economic losses, which causes haemorrhage and putrescence of hepatopancreas. To develop a more effective oral vaccine against IPNV infection, the aeromonas hydrophila adhesion (AHA1) gene was used as a targeting molecule for intestinal epithelial cells. A genetically engineered Lactobacillus casei (pPG-612-AHA1-CK6-VP2/L. casei 393) was constructed to express the AHA1-CK6-VP2 fusion protein. The expression of interest protein was confirmed by western blotting and the immunogenicity of pPG-612-AHA1-CK6-VP2/L. casei 393 was evaluated. And the results showed that more pPG-612-AHA1-CK6-VP2/L. casei 393 were found in the intestinal mucosal surface of the immunized group. The Lactobacillus-derived AHA1-CK6-VP2 fusion protein could induce the production of serum IgM and skin mucus IgT specific for IPNV with neutralizing activity in rainbow trouts. The levels of IL-1β, IL-8 and TNF-α isolated from the lymphocytes stimulated by AHA1-CK6-EGFP produced were significantly higher than EGFP group. For transcription levels of IL-1β, IL-8, CK6, MHC-II, Mx and TNF-1α in the spleen, the result indicated that the adhesion and target chemokine recruit more immune cells to induce cellular immunity. The level of IPNV in the immunized group of pPG-612-AHA1-CK6-VP2/L. casei 393 was significantly lower than that in the control groups. These data indicated that the adhesion and target chemokine could enhance antigen delivery efficiency, which provides a valuable strategy for the development of IPNV recombination Lactobacillus casei oral vaccine in the future.

The effects of optical genetic techniques on new neurons through the Wnt/β-Catenin pathway

To investigate the effects of optical genetic techniques on new neurons through the Wnt/β-Catenin pathway. Neural stem cells (ESCs)were extracted from the cerebral cortex of fetal rat and transfected by lentivirus carrying DCX-ChR2-EGFP gene and the expression of DCX of newborn neurons differentiated from neural stem cells were observed. All cells were divided into 3 groups(n=9): control group, NSCs+EGFP and NSCs+ChR2 groups. The control group was normal cultured NSCs (NSCs group); the neural stem cells in NSCs+EGFP group were transfected with lentivirus carrying EGFP gene. The neural stem cells in NSCs+ChR2 group were infected with lentivirus carrying DCX-ChR2-EGFP gene. After 48 hours of lentivirus infection, 470 nm blue laser irradiation was performed for 3 consecutive days. NeuN+ positive cell density(the maturation of neural stem cells)and the ratio of NeuN+/Hoechst in each group were observed. Western blot was used to detect the expression levels of MAP2, NeuN, Neurog2, NeuroD1 and GluR2. Western blot was used to detect the expressions of β-catenin and TCF4 associated with Wnt/β-catenin signaling channel. Verapamil (100 μmol/L, L-type calcium channel blockers) and Dkk1 (50 μg/ml, β-catenin inhibitor) were used to treat stem cells of the NSCs+ChR2 group and then the expressions of MAP2, NeuN, Neurog2, NeuroD1 and GluR were detected by Western blot. After 3 days of 470 nm blue laser irradiation, NeuN+ positive cell density(the maturation of neural stem cells)and the ratio of NeuN+/Hoechst, the expression levels of the protein MAP2, NeuN, Neurog2, NeuroD1, GluR and the protein β-catenin and TCF4 associated with Wnt/β-catenin signaling channel detected by Western blot were significantly increased in the group of NSCs+ChR2, compared with NSCs and NSCs+EGFP groups. The expressions of MAP2, NeuN, Neurog2, NeuroD1 and GluR were remarkably decreased after treated by verapamil and Dkk1 in the group of NSCs+ChR2. It was proved that the opening of ChR2 channel producing cationic influx promoted the maturation of neural stem cells and induced by the Wnt/β-catenin signaling pathway. Optical genetic promoted the maturation of newborn neurons through the Wnt/β-catenin signaling pathway.

Overexpression of LRIG1 gene enhances chemosensitvity of human bladder cancer T24 cell line to cisplatin

Objective To investigate the effects and mechanisms of LRIG1 overexpression on chemosensitivity of human bladder cancer T24 cell line to cisplatin. Methods Human bladder cancer T24 cell line was used in the experiment since December 2012. Firstly, human bladder cancer T24 cells which were capable of highly expressing exogenous LRIG1 gene were established and identified. The T24 cells were divided into 3 groups: experimental group (transfected with Ad–Surp–LRIGl), control group (transfected with Adeno–SP–EGFP), blank control group (untransfected). Secondly, experiments of CDDP–induced apoptosis were divided into 4 groups: control group (untreated), CDDP group (treated with CDDP), CDDP/Adeno–SP–EGFP group (treated with CDDP/Adeno–SP–EGFP and CDDP), CDDP/Ad–Surp–LRIG1 group (treated with CDDP/Ad–Surp–LRIG1 and CDDP). The expression of mRNA and protein of LRIG1 and EGFR were detected by RT–PCR and Western blot. The effect of LRIG1 and/or CDDP on the expression of EGFR and pEGFR of T24 cells were measured by Western blot. The cellular sensitivity to CDDP was evaluated by cell counting Kit–8 (CCK–8). The cells apoptosis rates were examined by flow cytometry. Results The mRNA expression of LRIG1 and EGFR in experimental group, control group and blank control group were (2.79±0.15) and (0.65±0.05), (0.76±0.09) and (1.98±0.07), (0.66±0.05) and (2.05±0.04); the protein expression of LRIG1 and EGFR in experimental group, control group and blank control group were(1.98±0.12) and (0.92±0.05), (0.88±0.07) and (2.51±0.07), (1.00±0.08) and (2.42±0.09). The differences were significant between experimental group and the other groups(P 0.05). The IC50 values in CDDP group, CDDP/Adeno–SP–EGFP group and CDDP/Ad–Surp–LRIGl group were (30.96±0.57) μg/ml, (31.55±0.48) μg/ml and (14.09±0.31) μg/ml. The difference were significant between CDDP/Ad–Surp–LRIGl group and the other groups(P 0.05). The apoptosis rate in CDDP/Ad–Surp–LRIGl group [(69.77±2.14)%] was significantly higher than that in CDDP/Adeno–SP–EGFP group [(48.15±1.53)%], CDDP group [(46.82±1.23)%] and control group [(3.17±0.21)%] (P 0.05). The expression of total EGFR protein and nuclear pEGFR protein in control group、CDDP group、CDDP/Adeno–SP–EGFP group, CDDP/Ad–Surp–LRIGl group were (2.52±0.11) and (1.76±0.08), (1.22±0.05) and (2.75±0.15), (1.25±0.05) and (2.82±0.13), (0.32±0.02) and (1.08±0.04). The differences between CDDP group and control group, CDDP/Adeno–SP–EGFP group and CDDP/Ad–Surp–LRIGl group were significant(P 0.05). Conclusions LRIG1 promotes the cisplatin–induced apoptosis of T24 cells and enhances the sensitivity to cisplatin by down–regulating the expression of EGFR and pEGFR. Key words: Leucine–rich repeats and immunoglobulin–like domains–1 gene; Cis–diaminodichloroplatinum; Bladder neoplasm; Apoptosis

Effect of the combined use of oncolytic adenovirus carrying tumor necrosis factor-related apoptosis-inducing ligand with epirubicin on proliferation and apoptosis of bladder cancer T24 cells

Objective To observe the effects of the combined use of oncolytic adenovirus carrying tumor necrosis factor-related apoptosis- inducing ligand(ZD55-TRAIL) with epirubicin(EPB) on the growth and apoptosis of bladder cancer T24 cells. Methods T24 cells were divided into 5 groups: ZD55-TRAIL[multiple of infection(MOI)= 10.0] plus EPB(1 mg/L) group, ZD55- TRAIL(MOI= 10.0) group, EPB(1 mg/L)group, ZD55- enhanced green fluorescent protein(EGFP, MOI= 10.0)group, and PBS group. The expression of TRAIL was detected by reverse transcriptase- polymerase chain reaction(RT - PCR)and Western blotting. The cytotoxicity of cells was assessed by cell counting kit- 8(CCK- 8)assay. Cell apoptosis was determined by annexin V- fluoresceine isothiocyanate(FITC)assay. Results RT-PCR analysis confirmed that T24 cells treated with ZD55- TRAIL plus EPB and ZD55- TRAIL could express TRAIL mRNA with high efficiency and there was no significant difference between two groups(P> 0.05). Western blotting analysis showed that ZD55- TRAIL could effectively promote the expression of TRAIL protein in T24 cells.CCK- 8 assay revealed the survival rate after 48 h in the ZD55- TRAIL(MOI= 10.0)plus EPB(1 mg/L)group[(18.44±2.38)%] was significantly higher than in ZD55- TRAIL group[(40.75± 4.12)%], EPB group[(53.62±1.41)%]and ZD55- EGFP group[(61.65±4.53)%,P<0.01]. The single use of EPBat the 2nd day achieved the same effect as MOI= 10.0 ZD55- TRAIL+ 1 mg/L EPBat 2 mg/ L,and at 3rd day,a higher concentration of 4 mg/L was needed. The results of cell apoptosis revealed the number of apoptosis cells in ZD55- TRAIL plus EPB group was 2.01 times,2.21 times, and 2.95 times of that in ZD55- TRAIL group, EPB group and ZD55- EGFP group respectively(P<0.01). Conclusion ZD55 - TRAIL conjugated with EPB exhibited a remarkably increased apoptosis- inducing effect and great antitumor effects in bladder cancer T24 cells. Key words: Oncolytic adenovirus; Tumor necrosis factor-related apoptosis-inducing ligand; Epirubicin

GW24-e1111 The effects of ACE2 on RAS system by painting on epicadial in atrial rapid pacing canine

ObjectivesTo study the effects of ACE2 on RAS system in rapid atrial pacing canine, and to investigate the possible mechanisms of ACE2 in improving atrial fibrillation "matrix" by inhibiting RAS...

IMPACT OF HUMAN HCN4 AND TBX3 GENE CO-TRANSFECTED LENTIVIRUS ON BIOLOGICAL ACTIVITY OF EMBRYONIC STEM CELL (ES)

ObjectivesT-box3 (Tbx3) is a family member of the T-box (Tbx) gene, which is a family of transcription factors involved in the regulation of developmental processes. The previous study showed that...

The Integration and Functional Evaluation of Rabbit Pacing Cells Transplanted into the Left Ventricular Free Wall

To evaluate the feasibility of cell transplantation to treat bradyarrhythmia, we analyzed the in vivo integration and pacing function after transplantation of mHCN4-modified rabbit bone marrow mesenchymal stem cells (MSCs) into the rabbit left ventricle free wall epicardium. In our investigation, we injected MSCs transduced with or without mHCN4 into the rabbit left ventricle free wall epicardium. Chemical ablation of the sinoatrial node was performed and bilateral vagus nerves were sequentially stimulated to observe premature left ventricular contraction or left ventricular rhythm. We found that the mHCN4-transduced MSC group had a significantly higher ventricular rate and a shorter QRS duration than that of the control and EGFP group. Furthermore, the mHCN4-transduced MSCs, but not the control cells, gradually adapted long-spindle morphology and became indistinguishable from adjacent ventricle myocytes. The modified MSCs showed pacing function approximately 1 week after transplantation and persisted at least 4 weeks after transplantation. In conclusion, a bradyarrhythmia model can be successfully established by chemical ablation of the sinoatrial node and sequential bilateral vagus nerve stimulation. The mHCN4-modified rabbit MSCs displayed evident dynamic morphology changes after being transplanted into rabbit left ventricle free wall epicardium. Our studies may provide a promising strategy of using modified stem cell transplantation to treat bradyarrhythmia.

Protective functions of recombinant protein targeted at RANKL against hepatic ischemia/reperfusion injury transfected by retrovirus in mice

To explore the protective functions of recombinant protein RANK-Fc against hepatic ischemia/reperfusion injury and clarify its possible mechanism. Sixty male Balb/c mice were randomly divided into 3 groups according to different treatments: serum-free medium control (Sham) group, target gene retrovirus (RANK-Fc) group and empty vector retrovirus (eGFP) group. All mice were injected with 2.5 ml solution (with or without retrovirus) within 6 seconds via tail vein. After 3 days, the model of 70% hepatic ischemia/reperfusion was induced under warm conditions for 90 minutes after different periods of reperfusion in RANK-Fc and eGFP groups; Sham group underwent the same procedure without the occlusion of blood supply. Blood and liver samples were obtained at different time points (1, 3, 6 and 24 h; n = 5 in each). Reverse transcription-polymerase chain reaction (RT-PCR) was used for the evaluation of eGFP mRNA expression. RANK-Fc was assessed by Western blot. Liver transaminases and histopathological changes were used for the evaluation of hepatic injury. The activity of NF-κB in liver nucleus was analyzed by Western blot and immunohistochemistry. The activation level of JNK was also assessed by Western blot. Liver homogenate levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 were detected by enzyme-linked immunosorbent assay (ELISA). Apoptosis was identified by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis. The differences between three treatment groups at each time point were detected by the one-way ANOVA. Statistical analysis for inter-comparison was performed by Student's t test. RANK-Fc and eGFP were successfully expressed in liver after hydrodynamics-based transfection. Compared with eGFP group, RANK-Fc significantly improved liver functions at the same time point (P < 0.01), decreased NF-κB nuclear translocation (t = 6.726, P < 0.01)and JNK phosphorylation (t = 6.713, P < 0.01)and obviously suppressed the release of pro-inflammatory cytokine TNF-α (t = 4.779, P < 0.01) and IL-6 (t = 5.482, P < 0.01). Morphological injuries were markedly alleviated while the expressions of immunohistochemical positive cells and TUNEL staining positive cells decreased in RANF-Fc group. RANK-Fc has protective functions against hepatic ischemia/reperfusion injury in mice. Its mechanism is at least partially related with the suppressions of proinflammatory NF-κB and proapoptotic JNK signaling pathways.

Allogeneic bone marrow mesenchymal stem cells over-expressing gap junction protein connexin 43 reduce ventricular arrhythmias following myocardial infarction in rats

BackgroundMesenchymal stem cell transplantation is a promising new therapy to improve cardiac function after myocardial infarction (MI). The electrophysiological consequences of MSC implantation have not been systematically studied.ObjectiveThis study strived...

Therapeutic effect of transplantation of bone marrow mesenchymal stem cells over-expressing Cx43 on heart failure in post-infarction rats

To explore the therapeutic effect of transplantation of bone marrow mesenchymal stem cells (BMSCs) over-expressing Cx43 on heart failure in post-infarction rats. Sixty SD rats were randomly divided equally into 4 groups: sham group, DMEM/F12 group injected with DMEM/F12, EGFP group with transplanted EGFP transfected BMSCs and Cx43 group with transplanted Cx43 transfected BMSCs. Myocardial infarction model was established by ligating anterior descending branch and the cells were transplanted after 30 minutes. At Week 4 post-infarction, the heart functions of rats were evaluated by echocardiography. After the rats were sacrificed, their tissue samples were collected. The areas of myocardial infarction and the levels of collagen fiber content were measured. And the expressions of EGFR and Cx43 were observed under laser confocal microscopy. The level of Cx43 mRNA was measured by reverse transcription-polymerase chain reaction (RT-PCR). As compared with the DMEM/F12 group, cardiac function was improved significantly, myocardial infarct area shrunk and collagen fiber content decreased significantly in the EGFP and group in Cx43 groups the. Survival of BMSCs and the formation of gap junction between BMSCs and the host myocardium could be observed under laser confocal microscopy both in EGFP group and Cx43 groups. And the post-infarction, expression of Cx43 mRNA in myocardial tissue decreased significantly in the group DMEM/F12, when compared with sham group (0.18 ± 0.05 vs 0.78 ± 0.14, P < 0.01). There was no significant difference on expression of Cx43 mRNA between DMEM/F12 group and EGFP group (0.18 ± 0.05 vs 0.20 ± 0.09, P > 0.05). The lever of Cx43 mRNA was higher in group Cx43 than in group DMEM/F12 and group EGFP(0.39 ± 0.14 vs 0.18 ± 0.05, P < 0.01; 0.39 ± 0.14 vs 0.20 ± 0.09, P < 0.05). Transplantation of BMSCs attenuates ventricular remodeling and improves cardiac functions. It may result from the over-expression of Cx43 gene through its effects of improving gap junction remodeling and increasing electro-mechanical coupling between myocardial cells in peri-infarct area.

The effects of human amniotic cells transfected with GDNF gene on hippocampal neuron following traumatic brain injury in rats

Objective To investigate the effect of grafting transfecting GDNF gene human amniotic cells (HACs) on hippocampal neuron following traumatic brain injury (TBI) in rats. Methods The model of hippocampal neuronal death following TBI was built up by improved Feeney's weight drop techniques. 5 μl transfecting HACs were injected into the margin of contusion 24 h after injured by microsyringe and stereotactic frame. Groups consisted of transfecting GDNF gene HACs group(TBI + GDNF), transfecting eGFP gene HACs group(TBI +eGFP), PBS group (TBI + PBS)and sham TBI group. The spatial memory performance was evaluated on 5 consecutive days by Morris water maze on 7 days after transplantation. After the probe trial, the rats were sacrificed for hippocampal neuron and brain tissue around target morphological analysis by cresyl violet staining under light microscopy. Expression of GDNF was detected by PCR in mRNA level. Results Transfecting GDNF gene HACs showed red fluorescence by immunofluorescence. The rats of TBI + GDNF group exhibited less escape latencies than those of TBI + eGFP group and TBI + PBS group, but had longer escape latencies than those of sham TBI group. Cresyl violet staining showed hippocampal neuronal loss, shrinkage and dark staining of neurons in TBI + eGFP group and TBI + PBS group. Compared with other groups, GDNF expression of TBI + GDNF group obviously increased in mRNA leveL Conclusion Grafting HACs transfected by GDNF gene plays a role in protecting against hippocampal neuronal death following TBI. Key words: Craniocerebral trauma; Human amniotic cells; Glial cell line - derived neurotrophic factor; Models,animal

Maxillary sinus floor elevation using a tissue engineered bone complex with BMP-2 gene modified bMSCs and a novel porous ceramic scaffold in rabbits

Objectives To study the effects of maxillary sinus floor elevation by a tissue engineered bone complex with bone morphogenetic protein-2 (BMP-2) gene modified bone marrow stromal cells (bMSCs) and a novel porous ceramic scaffold (OsteoBone™) in rabbits. Materials and methods bMSCs derived from New Zealand rabbit bone marrow were cultured and transduced with adenovirus AdBMP-2 and with AdEGFP gene (without BMP-2 gene sequence) as a control, respectively, in vitro. These bMSCs were then combined with OsteoBone™ scaffold at a concentration of 2 × 10 7 cells/ml and used to elevate the maxillary sinus floor in rabbits. Eight rabbits were randomly allocated into groups and sacrificed at weeks 2 and 4. For each time point, 8 maxillary sinus floor elevation surgeries were made bilaterally in 4 rabbits for the two groups ( n = 4 per group): group A (AdBMP-2-bMSCs/material) and group B (AdEGFP-bMSCs/material). All samples were evaluated by histologic and histomorphometric analysis. Results The augmented maxillary sinus height was maintained for both groups over the entire experimental period, while new bone area increased over time for group A. At week 4 after operation, bone area in group A was significantly more than that in group B ( P < 0.05), and was more obviously detected in the center of the elevated space. Under a confocal microscope, green fluorescence in newly formed bone was observed in the EGFP group, which suggests that those implanted bMSCs had contributed to the new bone formation. Conclusion bMSCs modified with AdBMP-2 gene can promote new bone formation in elevating the rabbit maxillary sinus. OsteoBone™ scaffold could be an ideal carrier for gene enhanced bone tissue engineering.

Recombinant lentivirus-mediated gene transfer of NT4-p53(N15)-Ant inhibits the growth of hepatocellular carcinoma cells in vitro

To construct a recombinant lentivirus vector containing fusion gene NT4-p53(N15)-Ant and transfer it into HepG2 cancer cells for gene therapy. The gene of p53(N15)-Ant was obtained by T-vector method. After restriction enzyme digestion, the interest gene of p53(N15)-Ant was inserted in pBV220/NT4 vector and fusion gene of NT4-p53(N15)-Ant was subcloned into the plasmid of lentivirus and cotransferred into HEK-293 cells with helper plasmid. The recombinant lentivirus was produced by homologous recombination of the above mentioned two plasmids in HEK-293 cells and its titer was measured by plaque-forming. The expression of LV. NT4-p53-Ant in transfected HepG2 cells was finally confirmed by reverse transcription polymerase chain reaction (RT-PCR) procedure. The effect of LV. NT4-p53(N15)-Ant on HepG2 cells was measured by a colorimetric 3-[4,5-dimethyl thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The inhibition effect on HepG2 cells of LV. NT4-p53(N15)-Ant and its potential mechanism was detected by light microscopy, electron microscopy, MTT, LDH-release assay and annexin V-PI double staining. The gene of p53(N15)-Ant was confirmed by restriction enzyme digestion and DNA sequencing. High titer of the recombinant lentivirus was obtained by homologous recombination in HEK-293 cell lines (1 x 10(11) pfu/ml), and the expression of NT4-p53(N15)-Ant gene in HepG2 cells was confirmed by RT-PCR. The viability of HepG2 cells was decreased to 83.4%, 46.9% and 33.9%, at 24 h, 48 h and 72 h, respectively, after infection by LV. NT4-p53(N15)-Ant. Compared with the LV. EGFP control group, there were significant differences (P < 0.01). The LDH level in HepG2 cells infected by LV. NT4-p53(N15)-Ant at 48 h, 72 h and 96 h after infection was 682 IU/L, 815 IU/L and 979 IU/L, respectively, significantly increased than that in the LV. EGFP group (P < 0.01), indicating the cell membrane destruction. The recombinant lentivirus vector encoding gene NT4-p53(N15)-Ant is successfully constructed in this experiment by molecular cloning and recombination in vitro techniques, and the results suggested that this fusion gene has an anti-tumor effect, which provides the basis for further research on recombinant adenovirus for cancer gene therapy.

Maxillary sinus floor elevation using a tissue‐engineered bone complex with β‐TCP and BMP‐2 gene‐modified bMSCs in rabbits

To study the effects of maxillary sinus floor elevation by a tissue-engineered bone complex with beta tricalcium phosphate (beta-TCP) and bone morphogenetic protein-2 (BMP-2) gene-modified bone marrow stromal cells (bMSCs) in rabbits. bMSCs derived from New Zealand rabbit bone marrow were cultured and transduced with the adenovirus with BMP-2 (AdBMP-2), adenovirus with enhanced green fluorescent protein gene (AdEGFP) in vitro. Gene transfer efficiency was detected by EGFP expression. These gene-modified autologous bMSCs were then combined with a beta-TCP granule scaffold at a concentration of 2 x 10(7) cells/ml and used to elevate the maxillary sinus floor in rabbits. Twenty rabbits were randomly allocated into groups and sacrificed at weeks 2 and 8. For each time point, 20 maxillary sinus floor elevation surgeries were made bilaterally in 10 rabbits for the following groups (n=5 per group): group A (beta-TCP alone), group B (untransduced bMSCs/beta-TCP), group C (AdEGFP-bMSCs/beta-TCP), and group D (AdBMP-2-bMSCs/beta-TCP). All samples were evaluated by histology and histomorphometric analysis. The fate of implanted bMSCs was traced initially by a confocol fluorescent microscope in the AdEGFP group. Gene transfer efficiency reached up to 60-80% with 50 PFU/cell transduction as demonstrated by fluorescent microscopic analysis in the AdEGFP group. The augmented maxillary sinus height was maintained for the four groups till 8 weeks post-surgery, while new bone area increased over the time. At week 2, bone areas in groups B-D were significantly larger than those in group A, while at week 8, in group D, the BMP-2 gene-enhanced tissue-engineered bone had the largest bone area among the groups (P<0.05, ANOVA). In that group, a mature bone structure was detected in the center of the elevated space. Under a confocal microscope, green fluorescence in newly formed bone was observed for the EGFP group, which suggested that those implanted bMSCs might have contributed to the new bone formation. bMSCs modified with the AdBMP-2 gene can promote new bone formation and maturation in the rabbit maxillary sinus. BMP-2 regional gene therapy and a tissue engineering technique could be effectively used in maxillary sinus elevation and bone regeneration.

Cardiac cholinergic NO-cGMP signaling following acute myocardial infarction and nNOS gene transfer

Myocardial infarction (MI) is associated with oxidative stress, which may cause cardiac autonomic impairment. We tested the hypothesis that acute MI disrupts cardiac cholinergic signaling by impairing nitric oxide (NO)-cGMP modulation of acetylcholine (ACh) release and whether the restoration of this pathway following cardiac neuronal NO synthase (nNOS) gene transfer had any bearing on the neural phenotype. Guinea pigs underwent four ligature coronary artery surgery (n = 50) under general anesthesia to induce MI or sham surgery (n = 32). In a separate group, at the time of MI surgery, adenovirus encoding nNOS (n = 29) or enhanced green fluorescent protein (eGFP; n = 30) was injected directly into the right atria, where the postganglionic cholinergic neurons reside. In vitro-evoked right atrial [3H]ACh release, right atrial NOS activity, and cGMP levels were measured at 3 days. Post-MI 24% of guinea pigs died compared with 9% in the sham-operated group. Evoked right atrial [3H]ACh release was significantly (P < 0.05) decreased in the MI group as was NOS activity and cGMP levels. Tetrahydrobiopterin levels were not significantly different between the sham and MI groups. Infarct sizes between gene-transferred groups were not significantly different. The nNOS transduced group had significantly increased right atrial [3H]ACh release, right atrial NOS activity, cGMP levels, and decreased cAMP levels. Fourteen percent of the nNOS transduced animals died compared with 31% mortality in the MI + eGFP group at 3 days. In conclusion, cardiac nNOS gene transfer partially restores the defective NO-cGMP cholinergic pathway post-MI, which was associated with a trend of improved survival at 3 days.

Protection of human islets from induction of apoptosis and improved islet function with HO-1 gene transduction

Islet transplantation represents an ideal therapeutic approach for treatment of type 1 diabetes but islet function and regeneration may be influenced by necrosis or apoptosis induced by oxidative stress and other insults. Heme oxygenase-1 (HO-1) is the rate-limiting enzyme in the catabolism of heme into biliverdin, releasing free iron and carbon monoxide. It has also been reported to be an antioxidant enzyme which can improve the function of grafted islets by cytoprotection via free radical scavenging and apoptosis prevention. In the present study, we investigated whether transduction of HO-1 genes into human islets with an adenovirus vector has cytoprotective action on islets cultured in vitro and discuss this method of gene therapy for clinical islet transplantation. Cadaveric pancreatic islets were isolated and purified in vitro. Transduction efficiency of islets was determined by infecting islets with adenovirus vector containing the enhanced green fluorescent protein gene (Ad-EGFP) at multiplicities of infection (MOI) of 2, 5, 10, or 20. Newly isolated islets were divided into three groups: EGFP group, islets transduced with Ad-EGFP using MOI = 20; HO-1 group, transduced with adenovirus vectors containing the human HO-1 gene using MOI = 20; and control group, mock transduced islets. Insulin release after glucose stimulation of the cell lines was determined by a radioimmunoassay kit and the stimulation index was calculated. Flow cytometry was used to detect apoptotic cells in the HO-1 group and in the control group after induction by recombinant human tumor necrosis factor-alpha (rTNFalpha) and cycloheximide (CHX) for 48 hours. Adenovirus vectors have a high efficiency of gene transduction into adult islet cells. Transduction of islets with the Ad-EGFP was most successful at MOI 20, at which MOI fluorescence was very intense on day 7 after transduction and EGFP was expressed in cultured islet cells for more than four weeks in vitro. The insulin release in the control group was (182.36 +/- 58.96) mIU/L after stimulation by high glucose media (16.7 mmol/L), while insulin release from the HO-1 group and the EGFP group were (270.09 +/- 89.37) mIU/L and (175.95 +/- 75.05) mIU/L respectively. Compared to the control group and the EGFP group, insulin release in the HO-1 group increased significantly (P < 0.05). After treatment with rTNFalpha and CHX the apoptotic ratio of islet cells was (63.09 +/- 10.86)% in the HO-1 group, significantly lower than (90.86 +/- 11.25)% in the control group (P < 0.05). Transduction of human islets with Ad-HO-1 can protect against TNF-alpha and CHX mediated cytotoxicity. The HO-1 gene also appears to facilitate insulin release from human islets. Transduction of donor islets with the adenovirus vector containing an HO-1 gene might have potential value in clinical islet transplantation.