lipofectamine and Disease-Models--Animal

Without effective medical interventions for complete reverse of NAFLD, it needs to urgently explore the underlying molecular mechanisms of non-alcoholic fatty liver disease (NAFLD) to offer a novel therapeutic strategy for people suffering from NAFLD. Sprague-Dawley (SD) rats were used to establish the NAFLD animal model. Lipofectamine 2000 was used to silence or over-express NEAT1. The expression of NEAT1 and the mRNA levels of ACC and FAS were determined by qRT-PCR. Western blot assays were performed to detect the expression of ACC and FAS at protein levels and the related protein levels of mTOR/S6K1 signaling pathway. The levels of liver triglyceride (TG), serum total cholesterol (TC), ALT, and AST were assessed by an automatic biochemistry analyzer. The levels of liver TG and serum cholesterol were obviously up-regulated in NAFLD rat model. The level of NEAT1 expression and the mRNA levels of ACC and FAS were obviously enhanced in NAFLD model both in vivo and in vitro. Knockdown of NEAT1 could also reduce the elevation of ACC and FAS induced by FFA in liver cells. Moreover, inhibition of mTOR/S6K1 pathway presented with the same effect with knockdown of NEAT1 on the expression of ACC and FAS mRNA levels. The injection of si-NEAT1 lentivirus was performed to treat NAFLD of rats and the obvious efficacy for NAFLD rats was achieved. In a word, the down-regulated level of NEAT1 could remit the non-alcoholic fatty liver disease through mTOR/S6K1 signaling pathway in rats.

Topics: Acetyl-CoA Carboxylase; Animals; Cell Line; Cholesterol; Disease Models, Animal; fas Receptor; Gene Knockdown Techniques; Humans; Lipids; Male; Non-alcoholic Fatty Liver Disease; Rats; Rats, Sprague-Dawley; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Long Noncoding; Signal Transduction; TOR Serine-Threonine Kinases; Up-Regulation

An efficient gene carrier to the brain is required for successful gene therapy of ischemic stroke. In this study, deoxycholic acid-conjugated polyethylenimine (DA-PEI) was synthesized and evaluated as a heme oxygenase-1 (HO-1) gene carrier for ischemic stroke gene therapy. Gel retardation assay and heparin competition assay showed that DA-PEI formed a stable complex with plasmid DNA. In vitro transfection assays with the luciferase gene showed that DA-PEI had higher transfection efficiency than polyethylenimine (25 kDa, PEI25k) and lipofectamine in Neuro2A cells. Furthermore, DA-PEI had less toxicity than lipofectamine. To evaluate the therapeutic effects of the pβ-HO-1/DA-PEI complex, the complex was injected locally in the brain of the transient middle cerebral artery occlusion animal model. In in vivo studies, DA-PEI was more effective than PEI25k in delivering pβ-HO-1 to the ischemic brain and achieved higher HO-1 expression. As a result, the pβ-HO-1/DA-PEI complexes more effectively reduced infarct volume and the number of apoptotic cells compared with the pβ-HO-1/PEI25k complex. The results suggest that DA-PEI will be useful for HO-1 gene therapy of ischemic stroke.

Topics: Animals; Apoptosis; Brain; Brain Ischemia; Cell Line, Tumor; Deoxycholic Acid; Disease Models, Animal; Gene Transfer Techniques; Genetic Therapy; Heme Oxygenase-1; Humans; Lipids; Male; Mice; Plasmids; Polyethyleneimine; Rats; Rats, Sprague-Dawley; Stroke; Transfection

In recent times, studies have demonstrated that carbon nanotubes are good candidates for use as vehicles for transfection of exogenous material into the cells. However, there are few studies evaluating the behavior of carbon nanotubes as DNA vectors and few of these studies have used multi-walled carbon nanotubes (MWCNTs) or carboxylated MWCNTs. Thus, this study aims to assess the MWCNTs' (carboxylated or not) efficiency in the increase in expression of the tetravalent vaccine candidate (TVC) plasmid vector for dengue virus in vitro using Vero cells, and in vivo, through the intramuscular route, to evaluate the immunological response profile.. Multi-walled carbon nanotubes internalized by Vero cells, have been found in the cytoplasm and nucleus associated with the plasmid. However, it was not efficient to increase the messenger ribonucleic acid (mRNA) compared to the pure vaccine candidate associated with Lipofectamine(®) 2000. The in vivo experiments showed that the use of intramuscular injection of the TVC in combination with MWCNTs reduced the immune response compared to pure TVC, in a general way, although an increase was observed in the population of the antibody-producing B cells, as compared to pure TVC.. The results confirm the data found by other authors, which demonstrate the ability of nanotubes to penetrate target cells and reach both the cytoplasm and the cell nucleus. The cytotoxicity values are also in accordance with the literature, which range from 5 to 20 µg/mL. This has been found to be 10 µg/mL in this study. Although the expression levels are higher in cells that receive the pure TVC transfected using Lipofectamine(®) 2000, the nanotubes show an increase in B-cells producing antibodies.

Topics: Animals; Antibodies, Viral; B-Lymphocytes; Chlorocebus aethiops; Dengue; Dengue Vaccines; Dengue Virus; Disease Models, Animal; Female; Genetic Vectors; Humans; Lipids; Lymphocyte Count; Mice; Mice, Inbred BALB C; Nanotubes, Carbon; Plasmids; Transfection; Vaccination; Vero Cells

Hepatitis B virus (HBV) is a persistent pathogen that causes acute and chronic necroinflammatory liver disease and is attributable to ~1 million deaths per year. In the present study, a conventional murine model was introduced based on the hydrodynamic injection of engineered replication‑competent HBV DNA into the tail veins of C57BL/6 mice. In a previous study, nine in‑frame ATG (start) codons in the S open reading frame (S1‑S9) were analyzed. The highly conserved ATG S5 was mutated to ACG by T378C, which led to the substitution sM75T and inhibition of the production and secretion of the hepatitis B surface antigen (HBsAg), and subsequent inhibition of HBV replication. In the present study, T378C was introduced into the pAAV‑HBV1.3 plasmid and was confirmed to affect HBsAg production and secretion, and HBV replication in vivo, which was in agreement with the previous in vitro results. Furthermore, the murine model was improved by co‑injection of the replication‑competent HBV plasmid DNA with Lipofectamine 2000 (LP). In this model, LP not only significantly enhanced HBV replication in mice, but also upregulated the expression of HBsAg and the hepatitis B core antigen. The current modified murine model was superior to the conventional murine HBV model based on HBV challenge by hydrodynamic injection.

Topics: Animals; Cell Line, Tumor; Disease Models, Animal; Hepatitis B Core Antigens; Hepatitis B Surface Antigens; Hepatitis B virus; Hepatitis B, Chronic; Humans; Hydrodynamics; Injections; Kinetics; Lipids; Mice; Mice, Inbred C57BL; Mutation; Plasmids; Virus Replication

Silencing of genes using small interfering RNA (siRNA) is a recently developed strategy to regulate the synthesis of target molecules. Signal transducer and activator of transcription 6 (STAT6) is a nuclear transcription factor that mediates Th2-type immunity.. To elucidate the therapeutic potential of using siRNA to inhibit STAT6 in allergic reactions, we determined the nucleotide sequences of siRNA specific for STAT6.. The selected sequences of STAT6 siRNA specifically inhibited the generation of STAT6 synthesis in dermal fibroblasts and eotaxin (CCL11) production in response to IL-4/TNF-α in vitro. Local administration of STAT6 siRNA in vivo alleviated contact hypersensitivity responses to chemical haptens. This was accompanied by reduced local production of IL-4, IL-13, eotaxin (CCL11), TARC (CCL17) and MDC (CCL22). Similarly, consecutive intranasal instillation of STAT6 siRNA markedly inhibited inflammatory cellular infiltration of mucosal tissues in allergic rhinitis responses in association with reduced IL-4 and IL-5 production from regional lymph node cells. Immediate responses, such as sneezing and nasal rubbing behaviors, were also improved by STAT6 siRNA.. Local administration of STAT6 siRNA is thus a promising therapeutic strategy for both Th2-mediated cutaneous diseases and allergic rhinitis.

Topics: Animals; Base Sequence; Chemokine CCL11; Dermatitis, Contact; Disease Models, Animal; Fibroblasts; Gene Silencing; Humans; Hypersensitivity; Interleukin-4; Lipids; Mice; Mice, Inbred BALB C; Molecular Sequence Data; NIH 3T3 Cells; Rhinitis; RNA, Small Interfering; STAT6 Transcription Factor; Th2 Cells; Treatment Outcome; Tumor Necrosis Factor-alpha

A major problem when using cationic polymers for gene delivery is that transfection is strongly inhibited by the presence of serum. This shortcoming limits the application of cationic polymers for systematic gene delivery in vivo. Due to the shielding effect of heparin, heparin conjugation to cationic polymers may improve the in vivo gene transfection efficiency. In this study, the transfection efficiency of heparin-conjugated polyethylenimine (HCPEI) with a low molecular weight of 1800 Da was compared to the transfection efficiencies of polyethylenimine with a low molecular weight of 1800 Da (PEI1800), polyethylenimine with a high molecular weight of 25,000 Da (PEI25k), and Lipofectamine. The size of the HCPEI/plasmid DNA (pDNA) complex is approximately 250 nm. HCPEI has a proton-buffering effect and HCPEI/pDNA has higher blood compatibility and a lower cytotoxicity than PEI25k/pDNA and Lipofectamine/pDNA. For in vitro transfection of rabbit smooth muscle cells in serum-free medium, the transfection efficiency of HCPEI/pDNA was not significantly different from those of PEI25k/pDNA and Lipofectamine/pDNA. Importantly, in serum-containing medium, the transfection efficiency of HCPEI/pDNA was significantly higher than those of PEI25k/pDNA and Lipofectamine/pDNA. For vascular endothelial growth factor (VEGF) gene transfection to mouse ischemic limbs, HCPEI/pDNA exhibited significantly higher VEGF expression and more extensive neovascularization than PEI/pDNA and Lipofectamine/pDNA. Taken together, heparin conjugation to PEI improves the in vivo gene transfection efficiency of PEI.

Topics: Animals; Cell Survival; Cells, Cultured; Disease Models, Animal; DNA; Erythrocyte Aggregation; Female; Genetic Therapy; Heparin; Hindlimb; Ischemia; Lipids; Mice; Mice, Inbred C57BL; Molecular Weight; Muscle, Skeletal; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Neovascularization, Physiologic; Particle Size; Polyethyleneimine; Rabbits; Rats; Rats, Sprague-Dawley; Transfection; Vascular Endothelial Growth Factor A

Topics: Animals; Cells, Cultured; Disease Models, Animal; Genetic Therapy; Genetic Vectors; Lipids; Liver Failure, Acute; Male; Mice; Mice, Inbred BALB C; Plasmids; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA Interference; RNA, Small Interfering; Signal Transduction; Transfection

Familial adenomatous polyposis is a highly penetrant, autosomal dominant disease resulting from a germline mutation of the adenomatous polyposis coli gene. Besides colorectal polyps and cancer, more than 90 percent of familial adenomatous polyposis patients also develop duodenal polyposis with an approximately 5 percent lifetime risk of malignant transformation. Because adenomatous polyposis coli protein has a "gatekeeper role" in the adenoma-carcinoma sequence, replacing its function may reduce polyp formation. We studied the functional outcome of per-oral, liposome-mediated adenomatous polyposis coli gene replacement therapy in a multiple intestinal neoplasia mouse model.. Twenty multiple intestinal neoplasia mice, heterozygous for the human homologue adenomatous polyposis coli gene, were randomly assigned to three groups: no treatment (n = 8); control plasmid containing green fluorescence protein reporter gene (n = 6); and plasmid containing the full-length adenomatous polyposis coli gene (n = 6). For the adenomatous polyposis coli-treated and green fluorescence protein reporter gene-treated groups, each mouse received the appropriate plasmid complexed with liposome, administered twice per week by oral gavage regime. Treatment lasted four weeks and all animals were killed at the end of treatment period with harvesting of intestinal tissue for polyp number estimation.. There was a statistically significant 25 percent reduction in the total number of polyps in the adenomatous polyposis coli-treated (73.1 +/- 1.4) group compared with untreated control (97.8 +/- 5.3, P < 0.01, Tukey test) and multiple intestinal neoplasia mice treated with control green fluorescence protein gene (103.3 +/- 1.7, P < 0.01, Tukey test).. Adenomatous polyposis coli gene dysfunction underlies tumorigenesis in familial adenomatous polyposis patients and multiple intestinal neoplasia mice. This in vivo study provides evidence to support a novel anti-adenoma strategy using enteral adenomatous polyposis coli gene replacement therapy.

Topics: Adenomatous Polyposis Coli; Administration, Oral; Analysis of Variance; Animals; Disease Models, Animal; Enteral Nutrition; Gene Transfer Techniques; Genes, APC; Genes, Reporter; Genetic Therapy; Germ-Line Mutation; Green Fluorescent Proteins; Humans; Intestinal Neoplasms; Lipids; Mice; Mice, Inbred C57BL; Plasmids; Random Allocation; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Tissue Distribution; Treatment Outcome

Previous research has revealed an antinociceptive (analgesic) effect of interleukin-2 (IL-2) in central and peripheral nervous systems. Unfortunately IL-2 is very short-lived in vivo, so it is impractical to apply IL-2 for analgesia in clinic. This study was performed to evaluate the effect of intrathecal delivery of human IL-2 gene on rat chronic neuropathic pain induced by chronic constriction injury of the sciatic nerve. Human IL-2 cDNA was cloned into pcDNA3 containing a cytomegalovirus promoter. The paw-withdrawal latency induced by radiant heat was used to measure the pain threshold. The results showed that recombinant human IL-2 had a dose-dependent antinociceptive effect, but that this only lasted for 10-25 min. The pcDNA3-IL-2 or pcDNA3-IL-2/lipofectamine complex in contrast also showed dose-dependent antinociceptive effects, but these reached a peak at day 2-3 and were maintained for up to 6 days. Liposome-mediated pcDNA3-IL-2 produced a more powerful antinociceptive effect than pcDNA3-IL-2 alone. The paw-withdrawal latencies were not affected by control treatments such as vehicle, lipofectamine, pcDNA3, or pcDNA3-lipofectamine. In the experimental groups, human IL-2 mRNA was detected by reverse transcription-polymerase chain reaction in the lumbar spinal pia mater, dorsal root ganglion, sciatic nerve, and spinal dorsal horn, but not in gastrocnemius muscle. The expressed IL-2 profile detected by western blot coincided with its mRNA profile except it was present in the spinal dorsal horn at a higher level. Furthermore, human IL-2 assayed by enzyme-linked immunosorbent assay in cerebrospinal fluid could still be detected at day 6, but lower than day 3. The antinociceptive effect of pcDNA3-IL-2 could be blocked by naloxone, showing some relationship of the antinociceptive effect produced by IL-2 gene to the opioid receptors. It is hoped that the new delivery approach of a single intrathecal injection of the IL-2 gene described here may be of some practical use as a part of a gene therapy for treating neuropathic pain.

Topics: Animals; Cation Exchange Resins; Chronic Disease; COS Cells; Disease Models, Animal; DNA, Complementary; Dose-Response Relationship, Drug; Genetic Therapy; Humans; Injections, Spinal; Interleukin-2; Ligation; Lipids; Liposomes; Male; Naloxone; Narcotic Antagonists; Organ Specificity; Pain; Pain Management; Pain Measurement; Rats; Rats, Sprague-Dawley; Recombinant Proteins; RNA, Messenger; Sciatic Neuropathy; Signal Transduction; Tissue Distribution; Transfection