OBJECTIVE This paper aims to explore the new method of continuous delivery of epidermal growth factor to wounds by transfected fibroblasts to promote wound repair. METHODS It was constructed a novel chimeric expression plasmid in which the biologically active portion of the human epidermal growth factor (EGF) gene was fused in-frame to the human granulocyte colony-stimulating factor signal sequence. RESULTS Clonally selected human fibroblasts transfected with this construct could secrete biologically active EGF. After the transplantation of irradiated gene-transfected fibroblasts suspended in fibrin glue to murine full-thickness wounds, EGF could be demonstrated for at least seven days in the wounds, slowly decreasing from initially 470 ng/L to 140 ng/L in 7 days. No EGF was found in the wound at 14 days. CONCLUSION A single application of irradiated EGF gene transfected fibroblasts to wounds can continuously deliver the transgene in vivo and can be used to administer drugs to the wound bed during the crucial first seven days of wound-healing.
Objective To observe the permeability and stability of the transfection of antisense oligonucleotide (ASODN) hybridized epidermal growth factor receptor (EGFR) to retinal glial cells (RG).Methods Phosphorothioate and unmodified EGFR ASODN conjugated with 5′-isothioc yanate (5′-FITC) were encapsulated with or without lipofectin, and then added into human retinal glial cells culture media. The cellular permeability and stability of the transfection were observed by fluorescence microscopy in fixed cells.Results In the absence of lipofectin, phosphorothioate and unmodified EGFR ASODN were found in a few RG cells at 30 minutes, and in about 50% RG cells at 4 hours. Phosphorothioate EGFR ASODN were kept in RG cells for 3-4 hours and disappeared at about 8 hours. In the presence of lipofectin, phosphoro thioate and unmodified EGFR ASODN were found in a few RG cells at 15 minutes and about 70%-80% RG cells at 4 hours. Phosphorothioate EGFR ASODN were kept in cells for 10-12 hours, and phosphorothioate and unmodified EGFR ASODN were disapp eared at about 14 hours and 4 hours respectively.Conclusion 5′-FITC EGFR ASODN encapsulated with lipofectin could enter RG cells and express stably in RG cells. (Chin J Ocul Fundus Dis，2003，19：52-54)
ObjectiveTo investigate the bone regeneration potential of cell-tissue engineered bone constructed by human bone marrow mesenchymal stem cells (hBMSCs) expressing the transduced human bone morphogenetic protein 2 (hBMP-2) gene stably. MethodsThe full-length hBMP-2 gene was cloned from human muscle tissues by RT-PCR and connected into a vector to consturct a eukaryotic expression system. And then the gene expression system was transduced to hBMSCs with lipidosome. hBMSCs were transfected by hBMP-2 gene (experimental group) and by empty plasmid (negative control group), untransfected hBMP-2 served as blank control group. RT-PCR, dot-ELISA, immunohistochemical analysis and ALP activity were performed to compare and evaluate the situation of hBMP-2 expression and secretion after transfection. hBMSCs transfected by hBMP-2 gene were seeded on hydroxyapatite (HA) and incubated for 4 days to construct the hBMP-2 gene modified tissue engineered bone, and then the tissue engineered bone was observed by the inverted phase contrast microscope and scanning electron microscope. Then the hBMP-2 gene modified tissue engineered bone (group A, n=3), empty plasmid transfected hBMSCs seeded on HA (group B, n=3), hBMSCs suspension transfected by hBMP-2 gene (group C, n=3), and hBMP-2 plasmids and lipidosome (group D, n=3) were implanted into bilateral back muscles of nude mice. The osteogenic activity was detected by HE staining and alcian blue staining after 4 weeks. ResultsAt 48 hours and 3 weeks after transfection, RT-PCR and dot-ELISA results indicated that the transfected hBMSCs could express and secrete active and exogenous hBMP-2 stably. The immunohistochemical staining was positive, and the ALP activity in the transfected hBMSCs was significantly higher than that in two control groups (P < 0.05). The transfected hBMSCs had a good attaching and growing on the three-demension suface of HA under inverted phase contrast microscope and scanning electron microscope. In vivo study indicated that a lot of new bone formation was obviously found at 4 out of 6 sides of back muscles in group A. Some new bone formation at both sides of back muscles was observed in 1 of 3 mice in group B. No new bone formation was found in group C. A few new bone formation was observed at one side of back muscles in group D. ConclusionThe tissue engineered bone constructed by hBMP-2 gene modified hBMSCs and HA is able to express and secrete active hBMP2 stably and can promote new bone formation effectively in muscles of nude mice.
Objective To explore the effects of overexpression of human tissue inhibitors of metalloproteinase-1 (hTIMP-1) on proliferation of human liver cancer cell line HepG2 in vitro. Methods A recombinant adenoviral vector containing full-length cDNA of hTIMP-1 was generated and transfected into HepG2. The viral titer was checked by measuring GFP, and the expression of hTIMP-1 in vitro was detected by the techniques of Western blot and semi-quantitative RT-PCR. The ultrastructure was observed by transmission electron microscope and the effects of overexpression of hTIMP-1 on proliferation of HepG2 in vitro was analyzed by MTT assay and growth curve. Results The resultant AdhTIMP-1 was successfully constructed and the expression of hTIMP-1 was detected by Western blot and RT-PCR. The growth and proliferation of HepG2, which had been transfected with AdhTIMP-1, was significantly inhibited. Conclusion The proliferation of HepG2 was markedly inhibited by recombinant adenovirus-mediated overexpression of hTIMP-1, which may pave the way for further application in liver gene therapy.
Objective To review the current concepts of gene therapy approachesmediated by adenovirus vectors for bone trauma and bone disease. Methods The recent literature concerned gene therapy mediated by adenovirus vectors was reviewed, which provides new insights into the treatments of bone trauma and bone disease. Results Adenovirus vectors was efficient, achieved high expression after transduction, and could transfer genes to both replicating and nonreplicating cells, such as osteoblasts, osteoclasts, fibroblasts, chondrocytes, bone marrow stromal cells, etc. Gene therapy mediated by adenovirus vectors achieved affirmative results in enhancing bone union and in curing bone diseases, such as osteoporosis and rheumatoid arthritis. Conclusion Gene therapy mediatedby adenovirus offers an exciting avenue for treatment of bone trauma and bone diseases.
Objective To study the effect of transforming growth factor β1 (TGF-β1) plasmid on poly frosted-defrosted allogenic nerve transplantation. Methods Forty Wistar rats were randomly divided into two groups equally. A 2.0 cm sciatic nerve segment, 5 mm away from infrapiriformis muscle space, was removed and the defect was repaired with poly frosteddefrosted allogenic nerve. The TGF-β1 plasmids were injected into the nerve anastomosis and adjacent muscles in the experimental group, normal saline in the control group. The nerve specimens were sectioned for staining in the 6th and 12th weeks . Axonal count and statistical analyses were done. Results The grafted and distal nerve segments showed regenerated fibers in both groups. In the experimental group,less edema and more nerve fibers were observed in the 6th week. The grafted nerve segment was filled with regeneration axons, the myelinated nerve fibers arranged regularly, and the axons and the myelin sheaths developed well in the 12th week. There was significant difference in the number of regenerating axons between the experimental group 98.6±4.8／μm2 and control group 75.8±5.1／μm2 (Plt;0.01). Conclusion Multiple frost-defrost of allogenic nerve can reduce its antigenicity and increase itsusefulness in repairing nerve defects. Local use of TGF-β1 plasmid can enhance immunosuppression to reduce immuno rejection.
Objective To explore the effect of age and gene therapyon the differentiation of marrow mesenchymal stem cells （MSCs） of the rats. Methods MSCs from the young (1-month-old), adult (9-month-old), and the aged(24monthold) rats were expanded in culture and infected with adenovirus mediated human bone morphogenetic protein 2 gene (Ad-BMP-2). The expression of BMP-2 and osteoblastic markers such as alkaline phosphatase(ALP), collagen Ⅰ(Col Ⅰ), bone sialoprotein(BSP) and osteopontin(OPN) were assayed during the process of differentiation. Their abilities to induce ectopic bone formation in nude mice were also tested. Results There was no significant difference in the expression of BMP-2 among the 3 groups. ALP activity assay and semi-quantitative reverse transcription polymerase chain reaction(RT-PCR) demonstrated that there were no significant differences in the expression of osteoblastic markers ALP, Col-Ⅰ, OPN and BSP amongthe 3 groups. Histomorphometric analysis indicated that there were no significant differences in the volume of the newly formed ectopic bones in nude mice amongthe 3 groups. Conclusion MSCs obtained from the aged ratscan restore their osteogenic activity following human BMP-2 gene transduction, therefore provides an alternative to treating the aged bone disease.
Duchenne muscular dystrophy is an X-linked inherited progressive degenerative muscle disease caused by mutations in the dystrophin gene, and is one of the most common progressive muscular dystrophies. We will review the selection of genetic diagnosis methods for Duchenne muscular dystrophy, the selection of experimental animal models, and treatment for the primary cause (including gene replacement therapy, exon skipping therapy, genome editing, stop codon read-through therapy, and stem cell therapy), the treatment of secondary pathological reactions and methods of assessing disease progression. The purpose is to enrich clinicians’ knowledge of the disease and provide a reference and help for the clinical diagnosis and treatment of Duchenne muscular dystrophy.
The application of gene therapy in ocular diseases is gradually expanding from mono-gene inherited diseases to multigene, multifactorial, common and chronic diseases. This emerging therapeutic approach is still in the early exploratory stage of treating diseases, and the expected benefits and risks remain highly uncertain. In the delivery process of gene therapy drugs, viral vector is currently one of the most mature and widely used vectors. The occurrence of vector-associated immunity will affect the short-term and long-term effects of gene therapy, and even cause permanent and serious damage to visual function. Therefore, gene therapy vector-associated immunity is the focus and challenge for the safety and long-term efficacy of gene therapy. During the perioperative and follow-up of gene therapy, attention should be paid to the monitoring of vector-associated immune inflammation, and appropriate measures should be taken to deal with the corresponding immune response, so as to achieve the best visual benefits for patients.
【Abstract】ObjectiveSome studies have demonstrated that recombinant adenoviruses are efficient vectors for gene transfer to the venous wall and AdCMV.tk encoded thymidine kinase can be used to reduce restenosis. In this study AdCMV.tk was apply to human vein smooth muscle cells (SMC) and organ cultured saphenous veins to study its effects on proliferation of SMCs and reduction of intimal hyperplasia. MethodsThe adenovirus vector transferred tk gene and mark gene lacZ to the SMC of human saphenous veins and organ cultured vein segments. Various concentrations ganciclovir (GCV) were contained in culture media. The efficiency of gene transfer was studied by using Xgal staining. The proliferation of SMC was monitored by the method of trypan blue exclusion. The bystander effect was observed by mixed cell culture. After vein segments treated by AdCMV.tk+GCV and cultured for 14 days, HE and VG staining were carried out and intimal thickness was analysis by computer image system. ResultsAdenovirus vector could infect saphenous vein SMC efficiently both in cultured SMCs and organ cultured vein segments. Gene expression sustained 14 d at least. The inhibition of SMCs proliferation in vitro was a positive correlation in GCV concentrations and the levels of tk expression. The proliferation of SMCs transfectered lacZ wasn’t restrained by GCV (P＜0.05). In mixed cell experiment there was at least 55% reduction in total cell number when as few as 10% of the cells express tk. Assessment of this “suicide gene strategy” in saphenous vein organ culture model demonstrated that veins treated with AdCMV.tk+GCV had a significant reduction at 14 days in the intimal thickness compared to control group (P＜0.01). ConclusionThe results suggest that adenovirusmediated gene transfer of tk along with GCV administration may be a useful strategy to treat the proliferation of intimal hyperplasia of transplanting saphenous veins. Bystander effects are amplified by AdCMV.tk/GCV gene therapy system.