ObjectiveTo investigate target gene therapy for hepatocellular carcinoma (HCC). MethodsHerpes simplex virus thymidine kinase (HSVTK) gene was inserted into the gene of AFP enhancer/ALB promoter with adenoassociated virus (AAV) plasmid (WAV2) as a carrier, and a hybrid plasmid pWAV2/AFPALB/HYTK was constructed. Besides, plasmid pEGFP1/AFPALB was also constructed. Two kinds of plasmids were transferred into AFP positive cells HepG2 and AFP negative cells 7721, SPC and 7901.ResultsIt was found that enhance green fluorescence protein could only be seen in AFP positive cells HepG2. 710 bp DNA was amplified only in AFP positive HepG2 cells.ConclusionPlasmid pWAV2/AFPALB/HYTK for HCC demonstrates specificity in vitro.
Objective To investigate if the course of intervertebral disc degeneration (IDD) is delayed by injecting lentivirus (Lv) vector carrying bone morphogenetic protein 2 (BMP-2) and inhibitor of differentiation 1 (Id1) genes directly into the nucleus pulposus. Methods Thirty-two New Zealand white rabbits, 2.0-2.5 kg in weight and 4 months in age, were used to establish the IDD models at L3, 4, L4, 5, and L5, 6 discs with annular puncture via transabdominal approach. Thirty rabbits with successful modeling were randomly divided into 5 groups, 6 rabbits every group. At 4 weeks after modeling, rabbits were injected with Lv-BMP-2 (group A), with Lv-BMP-2 and Lv-Id1 (group B), with Lv-Id1 (group C), with Lv-green fluorescent protein (group D), and with PBS (group E). At 2, 4, and 8 weeks after injection, T2-mapping MRI was performed on 2 rabbits each group to obtain the T2 values, and then subsequently the lumbar disc tissues were harvested to test the mRNA expressions and contents of collagen type II and proteoglycan by real-time fluorescent quantitative PCR and ELISA methods. Results T2-mapping MRI demonstrated that there was no significant difference in the T2 value between different groups at immediate and 2 weeks after injection (P>0.05). The T2 value of groups A and B was significantly higher than that of groups C, D, and E at 4 weeks after injection (P<0.05), but no significant difference was observed between group A and group B (P>0.05). The T2 value of group B was significantly higher than that of the other groups at 8 weeks after injection (P<0.05). The real-time fluorescent quantitative PCR and ELISA showed that the expressions and contents of collagen type II and proteoglycan in group B were significantly higher than those in the other groups at 2, 4, and 8 weeks after injection (P<0.05). Conclusion Combined application of Lv-BMP-2 and Lv-Id1 can delay IDD changes in rabbit IDD models.
Survivin-D53A (SVV-D53A) is a dominant-negative mutant survivin, which represents a potential promising target for cancer gene therapy. The present study was designed to determine whether SVV-D53A plasmid encapsuled by DOTAP: Chol liposome would have the anti-tumor activity against SPC-A1 lung adenocarcinoma, and to detect the possible mechanisms. In our experiment, SPC-A1 cells were transfected in vitro with SVV-D53A plasmid and examined for protein expression by Western blot, then flow cytometric analysis was used to detect apoptosis. SPC-A1 lung adenocarcinoma xenografts were established in vivo in the nude mice, which received the i.v. administrations of SVV-D53A plasmid/liposome complexes. After mice were sacrificed, the paraffin-embedded tumor tissue sections were used for proliferating cell nuclear antigen (PCNA) expression and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)assay. Compared with the control group, the mice treated with SVV-D53A plasmid had an obviously reduced tumor volume, with high level of apoptosis and decreased cell proliferation in tumor tissue. The research results proved that the administration of SVV-D53A plasmid resulted in significant inhibition of SPC-A1 cells both in vitro and in vivo. The functional mechanism is that the anti-tumor response causes and induces tumor cell apoptosis.
Objective To study the effect of immunogene therapy on the improvement of the outcome of malignant tumor. Methods Literatures about immunogene therapy of malignant tumor were collected and reviewed. Results The major methods of immunogene therapy of malignant tumor were as follows： ①Transference of cytokin genes into lymphocytes. ②Genetically engineered autologous tumor cells used as vaccine. ③Combined gene therapy.Conclusion The therapeutic effect of malignant tumor can be improved by immunogene therapy.
Selective recognition of double strands DNA (dsDNA) has been a research hot spot in molecular biology and biomedicine for a couple decades. Based on the selective interaction between natural nucleic acid/synthetic molecular ligands and dsDNA, gene diagnosis, gene therapy and gene editing would be realized. Hairpin oligopolyamide is a molecular ligand with excellent cellular permeability and nucleases-resistance which can target dsDNA sequence with high affinity and specificity at minor groove. This paper reviews the binding properties and biomedical applications of hairpin oligopolyamide targeting dsDNA, which provide references for further design and application of hairpin oligopolyamide.
Objective To investigate the effect and mechanism of calcitonin gene-related peptide (CGRP) on the prevention and treatment of transplant vein graft disease. Methods The 25 New Zealand white rabbits were divided into three groups: an experimental group [n=8, the rabbit jugular veins transfected with adeno-associated virus vector tipe 2/1 containing CGRP gene (AAV2/1-CGRP)], a carrier group [n=9, transfected with mosaic adeno-associated virus vector tipe 2/1 containing LacZ gene (AAV2/1-LacZ)] and a control group (n=8, saline) and then the cervical veins were implantated into the ipsilateral carotid artery by reverse end-side anastomosis. At 4 weeks after surgery, the pathology of the specimens, CD68 immunohistochemistry, in situ β-galactosidase staining were obtained. The expression of CGRP gene was detected by reverse transcription-polymerase chain reaction (RT-PCR). Monocyte chemoattractant protein-1(MCP-1), tumour necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS) and matrix metalloproteinase-9 (MMP-9) were detected by real-time polymerase chain reaction (real-time PCR). Results The CGRP and LacZ gene expression was positive at postoperative 4 weeks. The intima/media ratio was significantly inhibited in the experimental group. Macrophage infiltration and expression of inflammatory mediators including MCP-1, TNF-α, iNOS and MMP-9 were also significantly inhibited in the experimental group. Conclusion Transfection of AAV2/1-CGRP inhibits inflammatory mediator expression, macrophage infiltration and neointimal hyperplasia in experimental vein graft disease.
ObjectiveTo investigate the synergistic antitumor effects of ionizing radiation and the cytosine deaminase (CD)/5-flurocytosine (5-FC) system therapy in human pancreatic cancer cell.MethodsThe expression vector containing CD was transfected into the human pancreatic cancer cell line PC3. The clones were picked out after G418 selection. The CD gene integration and expression were confirmed by the RT-PCR. The cytotoxicity to the cells with or without CD and (or) ionizing radiation under the treatment with 5-FC was measured by the MTT assay. The clonogenic assay was used to investigate the radiosensitizing effect of 5-FC on the PC3 cells transfected or untransfected with CD gene.ResultsThe CD gene was stably expressed in the PC3 cells transfected with CD gene. The cytotoxic effect of 5-FC was superior on the PC3 cells transfected than that of untransfected with CD gene (P<0.05) and which were enhanced in combination with the ionizing radiation (P<0.05). The CD/5-FC enhanced the radiosensitivity of PC3 cells transfected with CD gene (P<0.05). The change in the radiosensitivity was quantified by calculating the sensitization enhancement ratio (SER) at the clinically relevant dose of 2 Gy. The SER was 1.5 in the PC3 cells transfected with CD gene by giving ionizing radiation of 2 Gy.ConclusionsCD/5-FC system is a potenial radiosensitizer in PC3 cells transfected with CD gene. Ionizing radiation and CD/5-FC system is more effective for killing effect of PC3 cells than ionizing radiation or CD/5-FC system alone.
Objective To summarize the research progress of microRNA (miRNA) and its non-viral vector in intervertebral disc degeneration (IDD) and to investigate the potential of non-viral vector delivery of miRNA in clinical application. Methods The related literature about the role of miRNA in IDD and its non-viral delivery system was reviewed and analyzed. Results MiRNA can regulate the related gene expression level and further participate in the pathophysiologic process in degenerated intervertebral disc, miRNA delivered by various non-viral vectors has obtained an ideal effect in some diseases. Conclusion MiRNA plays a great role in the cellular and molecular mechanisms of IDD, as a safe and effective strategy for gene therapy, non-viral vector provides new possibilities for IDD treated with miRNA.
With the development of molecular and cellar cardiology, gene therapy to cardiovascular disease has become the hot spot and the direction of study. Now, preclinical studies on ultrasound-mediated gene delivery (UMGD) in cardiovascular disease have achieved some success, but it is still hindered by a series of practical challenges for clinical translation. Even so, UMGD still holds the promise to cardiovascular disease in gene therapy for its non-invasiveness, accuracy, safety and ability to deliver multiple genes with repeated deliveries. In this review, we will focus on the basic principle, the current development, the future prospect and drawbacks of UMGD in the therapeutic applications of cardiovascular disease.
Objective To summarize the research progress of gene-based therapeutic angiogenesis in lower limb ischemia, so as to provide a new method for non-invasive treatment of lower limb ischemia. Method The literatures on studies of gene-based therapeutic angiogenesis in lower limb ischemia in recent years were read and reviewed. Results The incidence of peripheral arterial disease had been increasing annually. How to effectively reduce the amputation rate and mortality rate of patients with critical limb ischemia was still a clinical problem that needs to be solved urgently. A large number of basic and clinical studies had shown that gene-based therapeutic angiogenesis could effectively induce angiogenesis and collateral circulation in ischemic tissue of lower limb, leading to the significant improvements of blood perfusion in ischemic areas. Additionally, the construction of many kinds of new non-viral gene delivery vectors could also improve the safety and effectiveness of gene therapy to a certain extent. Conclusion Although promising therapeutic effect of gene-based therapeutic angiogenesis brings new ideas and strategies for the treatment of lower limb ischemia, issues still exist that have not been solved.