ObjectiveTo investigate the effect of zinc finger protein A20 on lumbar intervertebral disc degeneration in rabbits.MethodsTwenty-six 3-month-old New Zealand rabbits, 2.0-2.5 kg in weight, were used to establish the model of intervertebral disc degeneration at L3, 4, L4, 5, and L5, 6 by transabdominal needle puncture. At 4 weeks after operation, the 24 rabbits were randomly divided into 4 groups after successful modeling, which checked by MRI. The target intervertebral discs of each group were injected with zinc finger protein A20 overexpressed adenovirus (Ov-A20 group), empty carrier adenovirus (NC group), phosphate buffer saline (control group), and shRNA-A20 adenovirus (Sh-A20 group). The biological responses of animals in each group were comprehensive scored before 1 day of injection and after 1, 2, 3, and 6 days of injection. At 2, 4, and 8 weeks after injection, the animals in each group were observed by MRI to obtain the exact T2 relaxation time (T2 signal value). After MRI examination, the animals were killed to take the degenerative intervertebral disc tissue; and the tissue was detected by Alcian blue staining to observed the intervertebral disc degeneration. The expressions of zinc finger protein A20, collagen Ⅱ, and aggrecan were detected by immunohistochemistry staining. The expressions of zinc finger protein A20, nuclear factor κB binding protein [P65, phosphate P65 (P-P65), collagen Ⅱ, aggrecan], inflammatory factors [tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β)], autophagy-related protein [LC3 (LC3Ⅱ/LC3Ⅰ) and P62] were detected by Western blot.ResultsThe comprehensive score of biological response in each group after injection was significantly lower than that before injection (P<0.05). At 6 days after injection, the comprehensive score of biological response in the Sh-A20 group was significantly lower than that in other groups (P<0.05), and there was no significant difference among other groups (P>0.05). The detection of MRI showed that the T2 signal value in the Ov-A20 group was the highest at 2, 4, and 8 weeks after injection (P<0.05), and the T2 signal value in the Sh-A20 group was the lowest at 2 and 4 weeks after injection (P<0.05). There was no significant difference between other groups (P>0.05). Alcian blue staining showed that the expression of aggrecan was the highest in Ov-A20 group and the lowest in Sh-A20 group at 4 weeks (P<0.05); the expression of aggrecan in Ov-A20 group was the highest at 8 weeks (P<0.05), and there was no significant difference between other groups (P>0.05). Immunohistochemical staining showed that the expressions of zinc finger protein A20, collagen Ⅱ, and aggrecan were the highest in Ov-A20 group and lowest in Sh-A20 group (P<0.05). Western blot showed that the expressions of zinc finger protein A20, collagen Ⅱ, aggrecan, and LC3 (LC3Ⅱ/LC3Ⅰ) proteins were the highest in the Ov-A20 group and the lowest in Sh-A20 group (P<0.05), while the expressions of P-P65, TNF-α, IL-1β, and P62 proteins were the lowest in Ov-A20 group and the highest in Sh-A20 group (P<0.05). There was no significant difference in the expression of p65 protein between groups (P>0.05).ConclusionZinc finger protein A20 can effectively regulate the process of lumbar intervertebral disc degeneration in rabbits by inhibiting inflammation.
Objective To explore the tumor suppressive effect of superantigen staphylococcal enterotoxin B (SEB) for mice beared by Lewis lung cancer cell (LLCC). Methods SEB gene expressive plasmid PCDH-SEB-GFP was constructed and transfected into LLCC and it was detected by Western blot. Tumor-bearing mice model was established by subcutaneous injection of LLCC and SEB expressive plasmid PCDH-SEB-GFP was successfully injected into the tumor to observe its suppressive effect on the growth of Lewis lung cancer in mice. Results SEB expression was detected after transfection of LLCC with SEB gene plasmid PCDH-SEB-GFP. Intratumoral injection of plasmid PCDH-SEB-GFP could significantly inhibit the growth of Lewis lung cancer in tumor-bearing mice. Conclusions Intratumoral injection of superantigen SEB expressive plasmid can inhibit the growth of Lewis lung cancer in mice. It deserves a further study that SEB gene can work as an exogenous antigen gene in immune gene therapy for lung cancer.
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 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 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.
ObjectiveTo construct a cationic microbubble (CMB), and investigate the enhancement of gene transfection efficiency and therapeutic effect of ultrasound-targeted microbubble destruction (UTMD) in vivo with CMB compared to definity MB (DMB).Methods In vitro, the CMB was prepared by the method of thin film hydration. The morphology, size, zeta potential, and gene-carrying capacity of CMB were compared with the DMB. In vivo, the firefly luciferase gene which was used as a reporter gene was targeted transfected into myocardium of 16 rats with CMB and DMB, respectively. The gene transfection efficiency and targeting were observed dynamically. Then, ischemia-reperfusion (I/R) model was performed on 64 rats. The models of 60 rats were successfully confirmed by using ultrasonography at 5 days after I/R. The rats were divided into 3 groups (n=20) randomly. The control group received DMB carrying empty plasmid for transfection; DMB group received DMB carrying AKT plasmid for transfection; and CMB group received CMB carrying AKT plasmid for transfection. The cardiac perfusion, cardiac function, infarct size, and infarct thickness were measured by ultrasonography and histological observations after treatment. In addition, the capillary and arteriolar densities were measured with immunohistochemical staining. The myocyte apoptosis was measured with TUNEL staining. The protein expressions of AKT, phospho-AKT (P-AKT), Survivin, and phospho-BAD (P-BAD) were measured by Western blot.ResultsThe size of CMB was uniformly. The zeta potential of CMB was significantly higher than that of DMB (t=28.680, P=0.000). The CMB bound more plasmid DNA than the DMB (P<0.05). The luciferase activity of myocardium were higher in CMB group than in DMB group bothin vitro and in vivo measurements (P<0.05). There was no significant difference between groups in the ratio of signal intensity in anterior wall to posterior wall, ejection fraction (EF), and fractional shortening (FS) at 5 days after I/R (P>0.05), but the above indexes were significant higher in CMB and DMB groups than in control group at 21 days after I/R (P<0.05). Besides, the above indexes were significant higher in CMB group than in DMB group at 21 days after I/R (P<0.05). The infarct size was the smallest and infarct thickness was the thickest in the CMB group, followed by DMB group, control group at 21 days after I/R. The capillary and arteriolar densities of CMB and DMB groups were significant higher than those of control group at 21 days after I/R (P<0.05). Besides, the capillary and arteriolar densities of CMB group were significant higher than those of DMB group (P<0.05). The apoptotic cells were the most in the control group, followed by DMB group, CMB group at 3 days after gene transfection, showing significant differences between groups (P<0.05). The protein expressions of AKT, P-AKT, Survivin, and P-BAD were significant higher in CMB and DMB groups than those in control group at 3 days after gene transfection (P<0.05). Besides, these protein expressions were significant higher in CMB group than those in DMB group (P<0.05).ConclusionThe DNA-carrying capacity and gene transfection efficiency are elevated by CMB, although its physicochemical property is the same as DMB. When ultrasound-targeted AKT gene transfection is used to treat myocardial I/R injury in rats, delivery of AKT with the CMB can result in higher transfection efficiency and greater cardiac functional improvements compared to the DMB.
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.
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 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.
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.