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 explore a new method of treating early avascular necrosis of femoral head (AVNFH). Methods Sixty-nine New Zealand adult rabbitswith a mean weight of 2.8 kg after AVNFH presenting were randomly divided into three groups. In group A, deproteinized bone(DPB) combined with the recombinant plasmid pcDNA3.1/vascular endothelial growth factor 165(VEGF165) was implanted in the drilled channel of the necrotic femoral head. In group B, only DPB was implanted. In group C, channel was drilled without DPB or plasmid implanted. Femoral head specimens were obtained 3 days, 1, 2, 4, 8 and 16 weeks after operation. The expression of VEGF165 was examined by RT-PCR, Western blot and immunohistochemical techniques. X-ray testedbone formation generally. Angiogenesis and repair of the femoral head were observed by histological and histomorphometric analysis. Results In group A, the expressions of VEGF165 mRNA and protein were detected 3 days postoperatively, reached apex 1 week and lasted more than 3 weeks after implantation. The ratios of IOD of collagen type Ⅰ were 0.29±0.11, 0.55±0.13 and 0.67±0.10 IOD/μm2 respectively at 2, 4 and 8 weeks postoperatively and the ratios of IOD of new capillary vessels were 0.33±0.10and 0.57±0.16 IOD/μm2 respectively at 2, 4 weeks postoperatively in group A, showing statistically significant difference (Plt;0.01) when compared with groups B and D. X-ray test indicated much bone callus formed early. Conclusion Transfection of the VEGF165 gene can enhance local angiogenesis at early stage andDPBVEGF165 compound can improve bone formation. Deproteinized bone combined with VEGF165 gene provides a potential method for therapy of osteonecrosis.
To study the expression of CTLA4Ig gene in diabetic rat and the effect of CTLA4Ig on longterm survival of the pancreatic islet. The rat pancreatic islet cell and muscle cell transfected with the cDNA for CTLA4Ig packaged with lipofectin vector. We examined the expression level of CTLA4Ig gene, T lymphocyte reaction and observed the expression of CTLA4Ig cDNA in diabetic rat and the action of CTLA4Ig in longterm survival of pancreatic islet transplanted and the transplanted rats. Results: The T lymphocyte reaction from peripheral intravenous blood at seventh day after surgery, the difference between two group was significant (Ｐ＜0.05). Only 2 out of 10 recipients of the experiment group (A) at the seventh day after pancreatic islet allograft had any detectable levels of CTLA4Ig, their concentration was 14 ng/ml, and 31 ng/ml. The average time of maintaining blood glycose in normal levels of the group A after pancreatic islets graft, 14.8±12.3 days, was significantly longer than 3.6±5.1 days of the control group (B) (Ｐ＜0.05). The average survival time of the group A, 24.0±10.8 days (the longest time was 45 days), was significantly longer than 11.8±4.8 days (the longest time was 21 days) of the group B (Ｐ＜0.01). Conclusions: The muclse cells and pancreatic islets of the recipient rat was transfected with CTLA4IgcDNA packaged with lipofectin, and CTLA4IgcDNA was expressed in recipient tissue, its expressed product CTLA4Ig make pancreatic islets transplanted and recipient rat survive longer significantly.
Fundus disease is a kind of common ophthalmic disease with high blindness rate and great harm. Although great breakthroughs have been made in medical treatment, laser photocoagulation, radiotherapy and gene therapy of fundus disease, with the further understanding of the essence of fundus disease, there are higher requirements for the treatment of fundus disease. To strengthen scientific research on the etiology, pathological mechanism and clinical treatment of fundus diseases, to control the quality of research, to develop reasonable treatment plans and explore new treatment methods under the guidance of evidence-based medicine theory, to further improve the level of medical treatment and benefit patients with fundus diseases will still be a formidable challenge in the future.
ObjectiveTo investigate the current status of research in gene therapy for retinitis pigmentosa (RP) from 2005 to 2024. MethodsThe literature related to gene therapy for RP included in the Web of Science Core Collection dataset from January 1, 2005 to September 15, 2024 was retrieved and screened. The bibliometrix package of R software was used to analyze the annual trend of the number of publications, citation frequency, distribution of countries/regions of the literature, and distribution of journals containing the articles. CiteSpace software was used to perform keyword clustering analysis and the keywords bursts analysis. ResultsA total of 209 articles were included. There was an overall fluctuating upward trend of annual publications from 2005 to 2024, with the highest number of publications in 2023 at 26 (12.4%, 26/209), and the lowest number of publications in 2006 at 2 (0.9%, 2/209). There was an overall increasing trend in the frequency of citations to relevant literature. Corresponding authors from the United States had the highest total number of publications with 98 (46.9%, 98/209). Among authors, Hauswirth from the University of Florida, USA, had the most with 25 (12.0%, 25/209). Among institutions, Columbia University, USA, had the most with 55 (26.3%, 55/209). Among journals, Mol Ther had the most with 25 (12.0%, 25/209), and it had the highest 2023 impact factor of 12.1. Keyword clustering analysis yielded eight valid clusters, namely #0 P23H, #1 AAV, #2 PDE6B, #3 CRB1, #4 RPGR, #5 antisense oligonucleotide, #6 NR2E3, and #7 NRL, which intersected with each other with good continuity. The keywords bursts analysis showed that the keyword with the longest emergence time was RNAi, followed by PDE and PDE6. USH2A, CRB1, CRISPR Cas9, base editing, and ORF15 were keywords that emerged in recent years and were continuously studied. ConclusionsRP gene therapy research literature has shown an increasing trend from 2005 to 2024, with the highest number of publications from research organizations and scholars in the United States. Currently, studies focus on RHO, PDE6B, CRB1, RPGR, NR2E3, and NRL gene. In recent years, there has been a gradual increase in studies on USH2A, CRB1 genes, and the RPGR ORF15 region. CRISPR Cas9 and base editing gene therapy strategies are being developed.
ObjectiveTo investigate the proliferation and apoptosis effects of adenovirus-mediated interleukin-24 (Ad-IL-24) gene on Karpas299 cells in vitro. MethodsThe Karpas299 cells were divided into blank control group, Ad-IL-24 group, and the adenovirus which carrying green fluorescent protein gene group (Ad-GFP group). Karpas299 cells of Ad-IL-24 group were infected by adding 200.0 μL Ad-IL-24, Karpas299 cells of Ad-GFP group were infected by adding 200.0 μL Ad-GFP, but Karpas299 cells of blank control group were treated by adding 200.0 μL PBS. Cells' proliferation inhibition rates of 3 groups were detected by cell counting kit (CCK-8) method at 12, 24, and 48 hours after treatment, respectively, and the cells' apoptosis rates of 3 groups were detected by flow cytometry at 48 hours after treatment. ResultsAd-IL-24 can suppress the growth of Karpas299 cells, and the inhibition rate increased over time. Compared with Ad-GFP group at the same time, the cell' proliferation inhibition rate of Ad-IL-24 group was higher at 12, 24, and 48 hours after treatment (P<0.05). In addition, the cells' apoptosis rate of Ad-IL-24 group was higher than those of Ad-GFP group and blank control group at 48 hours after treatment (P<0.05). ConclusionAd-IL-24 can suppress the growth of Karpas299 cells and induce the apoptosis of it.
【Abstract】Objective To construct a recombinant adenoviral vector carrying antisense matrix metalloproteinase2 (MMP2) for use in the gene therapy to inhibit the invasiveness and migratory capacity of hepatocellular carcinoma (HCC) cell line HepG2 in vitro and in vivo models. Methods Total RNA was extracted from HCC, and then a 500 bp fragment at the 5′ end of human MMP2 cDNA was synthesized by polymerase chain reaction (PCR) and was reversely inserted into the multiclone site (MCS) of the shuttle plasmid pAdTrack-CMV,with the resultant plasmid and the backbone plasmid pAdEasy-1,the homologous recombination took place in the E.coli BJ5183 and the recombinant adenoviral plasmid carrying the antisense MMP2 gene was constructed and generated. The adenoviruses(Ad-MMP2AS) were packaged and amplified in the HEK 293 cells.Then the viral titer was checked by GFP. Results The recombinant adenovirus vector carrying antisense MMP2 was constructed successfully, the b green fluorescence was observed in HEK 293 cells under a fluorescence microscopy. The viral titer was 1×108/ml. Conclusion The recombinant adenovirus Ad-MMP2AS constructed by us could introduce the antisense MMP2 into HepG2 effectively,which would provide experimental basis for reversing the overexpression of MMP2 in HCC and for inhibiting the invasiveness and migratory capacity of HepG2 in vitro and in vivo models.
Objective To explore the effect of Tie-2 small interference RNA (siRNA) treatment in human hepatoma transplanted subcutaneously in nude mice. Methods Tumor cells were implanted in the hind flank of male nude mice of 6 weeks. Tumor-bearing mice were divided into two groups （gene therapy group and control group） and injected intra-tumorally with Tie-2-siRNA/Lipofectamine and saline/Lipofectamine respectively. The tumor volume and weight, serum AFP and microvessel density (MVD) and the histological change of the tumor were tested after gene therapy. Results The growth inhibitory rates in gene therapy group were 26.94%， 53.01% and 68.91% on day 4， 7 and 10 after gene therapy respectively. The tumor volumes of gene therapy group (118.47, 111.57 and 104.59 mm3) were smaller than those of the control group (162.17, 237.46 and 336.41 mm3) respectively (P＜0.01), and the weight of tumor in gene therapy group was lighter than that of the control group 〔(0.89±0.09) g vs (1.24±0.03), P＜0.01〕. The AFP value in gene therapy group was obviously lower than that of the control group 〔(107.66±24.13) ng/ml vs (266.08±50.96) ng/ml, P＜0.01〕. There was significant diference of MVD between the gene therapy group (34.63±4.07) and the control group (81.01±9.44) with the method of immunohistochemitry (P＜0.01). Histopathology in the control group showed that the tumor volumes were bigger, and a high atypic of tumor cells were seen. The main pathological changes in tumor tissue of gene therapy group were necrosis, there were massive necrosis. The apoptosis cells were seen in the both of necrosis and non-necrosis areas in only 2 mice of gene therapy group. Conclusion Tie-2-siRNA inhibits the tumor growth and tumor angiogenesis， and is a possible new approach for liver neoplasm gene therapy.
Objective To study the vascularization of the compositeof bone morphogenetic protein 2 (BMP-2) gene transfected marrow mesenchymal stem cells (MSCs) and biodegradable scaffolds in repairing bone defect. Methods Adenovirus vector carrying BMP-2 (Ad-BMP-2) gene transfected MSCs and gene modified tissue engineered bone was constructed. The 1.5 cm radial defect models were made on 60 rabbits, which were evenly divided into 4 groups randomly(n=15, 30 sides). Different materials were used in 4 groups: Ad-BMP-2 transfected MSCs plus PLA/PCL (group A), AdLacz transfected MSCs plus PLA/PCL (group B), MSCs plus PLA/PCL (group C) and only PLA/PCL scaffolds (group D). The X-ray, capillary vessel ink infusion, histology, TEM, VEGF expression and microvacular density counting(MVD) were made 4, 8, and 12 weeks after operation. Results In group A after 4 weeks, foliated formed bones image was observed in the transplanted bones, new vessels grew into the bones, the pores of scaffolds were filled with cartilage callus, osteoblasts with active function grew around the microvessels, and VEGF expression and the number of microvessels were significantly superior to those of other groups, showing statistically significant difference (Plt;0.01); after 8 weeks, increasingly more new bones grew in the transplanted bones, microvessels distended and connected with each other, cartilage callus changed into trabecular bones; after 12 weeks, lamellar bone became successive, marrow cavity recanalized, microvessels showed orderly longitudinal arrangement. In groups B and C, the capability of bone formation was weak, the regeneration of blood vessels was slow, after 12 weeks, defects were mostly repaired, microvessels grew among the new trabecular bones. In group D, few new vessels were observed at each time, after 12 weeks, broken ends became hardened, the defectedarea was filled with fibrous tissue. Conclusion BMP-2 gene therapy, by -upregulating VEGF expression, indirectly induces vascularization ofgrafts,promotes the living of seed cells, and thus accelerates new bone formation.