Objective To introduce growth and differentiation factor 5 (GDF-5) gene into hBMSCs using recombinant adenovirus vector and to investigate the effect of GDF-5 gene expression on hBMSCs osteogenic differentiation. Methods Recombinant adenovirus GDF-5 (Ad-GDF-5) containing green fluorescent protein (GFP) and Ad-GFP were amplifiedand tittered. hBMSCs at passage 3 were infected with two viruses at different titers. At 2 days after intervention, GFP expression was observed using fluorescence microscope, and GDF-5 expression in hBMSCs was detected by RT-PCR. Adherent hBMSCs at passage 3 were randomly divided into 4 groups: experimental group (GDF-5 gene transfection), osteogenic induction group, Ad- GFP infection group, and control group. Cell differentiation was detected by inverted phase contrast microscope observation, fluorescence microscope observation, reverse transcription fluorescence quantitative PCR, immunofluorescence staining, and von Kossa staining at different time points after intervention. Results The titer of Ad-GDF-5 and Ad-GFP was 1.0 × 109 pfu/mL and 1.2 × 109 pfu/mL, respectively. hBMSCs was efficiently infected by Ad-GDF-5 and Ad-GFP, and expressed target gene and GFP gene. At 1-7 days after intervention, morphology and growth pattern of the hBMSCs in the experimental group and the osteogenic induction group were transformed into osteoblast-l ike cells, whereas the cells in the other two groups were still maintained their original morphology and growth pattern. Reverse transcription fluorescence quantitative PCR detection: at 4 days after intervention, GDF-5 expression in the experimental group was obviously higher than that of other groups (P lt; 0.05); ALP, Col I, and OC gene expression in the experimental and the osteogenic induction group were superior to those of theAd-GFP infection and the control group (P lt; 0.05); Col I gene expression in the osteogenic induction group was greater than that of the experimental group (P lt; 0.05). Immunofluorescence staining: at 4 days after intervention, the cells in the osteogenic induction group and the experimental group expressed and secreted Col I, and no expression of Col I was evident in the other two groups. At 10 days after intervention, the cells in the osteogenic induction and the experimental group were positive for von Kossa staining, and the results of the other two groups were negative. Conclusion GDF-5 gene can be transferred into hBMSCs via adenovirus vector and be expressed stably. It can facil itate the osteogenic differentiation of the hBMSCs and lay a foundation for the further study of this kind of gene transferred hBMSCs effect on bone tissue repair.
Objective To investigate the stable, high effective and low toxicity gene transfer vectors’ basics in research and clinical application.Methods Current status of hepatocytedirected gene transfer vectors were reviewed by history document and contemporary experimental advances analysis.Results Viral and nonviral vector systems could both transduce target genes to liver effectively with various transduction rates based on their corresponding biological characteristics.Conclusion Hepatocyte is the effective target for gene therapy of liverrelated genetic, neoplastic and infectious diseases, although the security needs to be further evaluated.
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.
ObjectiveTo investigate the expression of tumor suppressor gene Tat interacting protein 30 (TIP30) gene in papillary thyroid carcinoma and it’s clinical significance in treatment of thyroid carcinoma. Methods Thirty cases of pathological specimens wax pieces of papillary thyroid carcinoma from 2003 to 2006 in our hospital were selected, in which there were 7 male, 23 female； and the age was from 15 to 70 years old, average 44.7 years. Six cases were nodular goiter with carcinomatous change in local area (papillary), 2 cases were thyroid capsular invasion. Distant lymph node metastasis and lesions surrounding the thyroid tissue were not confirmed by pathology. Every specimen was divided into tumor tissue and adjacent tissue (1-2 cm far away from tumor and non-cancerous tissue was confirmed by pathology). The expression of TIP30 in specimen was detected by immunohistochemical method with staining index and the average absorbance. ResultsTIP30 was expressed in the cell membrane and cytoplasm, which was showed as brown particles. ①Staining index: TIP30 in adjacent tissues was expressed highly with 21 (70.0%) positive cases (gt;2 points) and 9 (30.0%) negative cases (≤2 points), while its expression in cancer tissues was reduced or missed with 11 (36.7%) positive cases (gt;2 points) and 19 (63.3%) negative cases (≤2 points). There was a statistical difference between them (P＜0.05), and it was not related to age and gender of patients (Pgt;0.05). ②The average absorbance of TIP30 in cancer tissues was significantly lower than that in adjacent tissue (P＜0.05). ConclusionThe expression of TIP30 in papillary thyroid carcinoma is reduced or deleted, which can supply some theory support for its gene therapy.
Objective To construct replication-defective adenovirus containing tk gene (ADV-tk). Methods Recombinant adenovirus of ADV-tk was constructed using homologous recombination in cells. After the interested tk gene fragment in the recombinant plasmid obtained was confirmed by PCR, the titre of purified recombinant adenovirus was detected. In vitro study, tk gene in SMMC7721 cells transfected by ADV-tk was investigated by RT-PCR. In vivo study, ADV-tk was injected intraperitoneally into BALB/c nude mice with liver cancer and apoptosis cells in tumor were observed. Results Recombinant adenovirus containing ADV-tk was proved successfully. The titre of purified recombinant adenovirus was 1.4×1010 pfu/ml. In vitro study, tk was integrated and expressed by SMMC-7721 cells. In vivo study, with the injection of ADV-tk, apoptosis cells in tumor increased. Conclusion A replication-defective adenovirus containing tk gene is successfully constructed, which may useful for further research on tumor suicide gene therapy with ADV-tk.
Retinitis pigmentosa is a hereditary disease which is characterized by damage in retinal photoreceptor cells and retinal pigment epithelium. Its main clinical features include low vision with night blindness, progressive visual field defects, and abnormal electroretinograms. The development of gene sequencing, the diagnosis and treatment methods of retinitis pigmentosa update year by year, including gene therapy, stem cell therapy, optogenetic therapy, etc. However, there is still a big gap in these treatments from laboratory technology into effective clinical treatment drugs. Some problems which include immune response, potential mutagenesis and tumorigenesis of the inserted region, genetic toxicity, quality and stability of gene technology and stem cell technology, mass production and promotion of clinical grade drugs, and optimization of the effectiveness of drugs and surgery, etc, remain to be solved by researchers.
Objective To construct the recombinant adenovirus bearing human transforming growth factor β1(TGF-β1) and bone morphogenetic protein 7 (BMP-7) genes, and investigate its co-expression in the marrow stromalstemcells (MSCs) and bioactivity effect. Methods Using the replication defective adenovirus AdEasy as a carrier, MSCs were infected by the high-titer-level recombinant adenovirus taking TGF-β1 and BMP-7 genes. Immunocytochemistry, in situ hybridization，reverse transcription-polymerase chain reaction (RT-PCR), and hexuronic acid level test were used to detect the coexpression of the exogenous genes and to analyze their effect transfection on directive differentiation of MSCs. Results The immunocytochemistry staining showed that the brown coarse grains were situated in the cytoplasm of the most MSCs 72 h after infection. Procollagen ⅡmRNA in the cells was detected by the in situ hybridization, and the content of hexuronic acid in the culture mediumwas significantly increased 10 days after infection compared with the level before infecton (Plt;0.01). Conclusion The recombinant adenovirus bearing human TGF-β1 and BMP-7 genes can be constructed, and the exogenous gene can be coexpressed in MSCs, which may offer a novel approach to thelocal combination gene therapy for repairing joint cartilage defects.
Objective To study the effects of pcDNA3/AFP/TK/Angio fusion gene targeting therapy for human primary liver cancer in nude mice implanted with SMMC-7721. Methods Human liver cancer cell line SMMC-7721 was implanted subcutaneously in nude mice to establish experiment model. Animals bearing liver cancer were randomly divided into five groups: control group， vector group， GCV (ganciclovir) group， pcDNA3/TK/Angio group; pcDNA3/AFP/TK/Angio group. Different plasmids were directly injected into tumors and GCV was intraperitoneally administrated simultaneously according to different groups. The growth of tumors was observed and the pathology was examined as well. Serum AFP level was measured by radioimmunology, the ultrastructural change of tumor cells was studied by using electron microscopy, the expressions of MVD and VEGF were respectively detected with immunohistochemistry and the cell apoptosis in situ was detected by TUNEL. Results The success rate to establish subcutaneous implanted liver cancer model in nude mice was 100%. The tumor volume, serum AFP level, VEGF and MVD expressions of pcDNA3/TK/Angio group and pcDNA3/AFP/TK/Angio group were lower than those in control group, vector group and GCV group (P＜0.05) and more apoptosis cells could be observed. While the tumor volume, serum AFP level, VEGF and MVD expressions of pcDNA3/AFP/TK/Angio group was lower than those in pcDNA3/TK/Angio group (P＜0.05); and apoptosis index was higher than that of the latter (P＜0.05).Conclusion pcDNA3/AFP/TK/Angio fusion gene inhibits the growth of tumor remarkably and becomes a promising new biological agent to treat human primary liver cancer.
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.
Objective To investigate the latest research and the therapeutic development in the injuries to the spine and spinal cord. Methods Literature concerned was reviewed, combined with our own research and clinical experience, to summarize the trend of the researches and their clinical application in the treatment of the injured spine and spinal cord.Results Theposterior approach atlantoaxial stabilization technique changed the conventional wiring technique into the transarticular screw fixation to the plate and pedicle or the lateral mass screw fixation technique. Theclinical application of the transoralpharyngeal atlantoaxial reduction plate fixation technique showed a good effect on the reduction of atlantoaxial dislocation. However, there were no unified criteria for selection of the surgical approach, fixation level, and fusion mode in the treatment of thoracolumbar spinalfractures. Under optimal conditions, both the anterior and the posterior approaches could achieve good clinical effects on decompression and spinal reconstruction. The single level fixation technique showed some advantages in treating certaintypes of thoracolumbar spinal fractures when compared with the traditional cross-sectional fixation. The endoscopy-assistant and image-guiding spinal intervention techniques were evolved in China during these years. In the treatment of the obstinate painful osteoporotic vertebral compressive fracture, percutaneous vertebroplasty and kyphoplasty achieved good results in the pain relief and spinal reconstruction. Numerous basic and clinical researches have given us a further understanding of the medical protection of acute spinal cord injury, and biological treatments have given us new ideas on neural reparation and regeneration. Cell transplantation and gene therapy have become the most promising treatment strategies in this field.Conclusion With the rapid development of spine surgery, the repair and reconstruction ofthe injured spine and spinal cord made a great stride in the recent years.
