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 explore the feasibility of recombinant adeno-associated virus (rAAV) as a vector for the gene therapy of liver cancer. Methods The rAAV/enhance green fluorescein protein (EGFP) recombinant was prepared by the routine method of two plasmids cotransfection.Results The experiment showed that one 10cm plate could produce 107-108 infection unit recombinant by the method of two plasmids cotransfection, and the transduction of HepG2 cell was increased with the increase of infection dosage of rAAV. About 100 multiplicity of infection (MOI) AAV vector could make all the tumor cell light. Conclusion Liver cancer cell can be efficiently transduced by rAAV, and AAV vector may be a valuable vector for the gene therapy of liver cancer.
OBJECTIVE Because of its special biological characteristics, myoblast might play a role in gene delivery and cell-to-biomaterial interactions. In this paper, the biological features of myoblast and its application on gene therapy and tissue engineering was discussed. METHODS Documents about proliferation and differentiation of myoblast were reviewed in details. The prospects of its application on gene therapy and tissue engineering were also presented. RESULTS Myoblast was important in muscle regeneration. The activation of myoblast to proliferate and differentiate was the very beginning of regeneration after injury. The cultured myoblast had high potential to proliferate, it was ready to fuse with each other and to form myotube (the special behavior of myoblast differentiation). Myoblast transplantation had been studied as a possible treatment for inherited myopathies, such as Duchenne muscular dystrophy. The transplanted myoblast could fuse with host myofibers, so the delivered target gene integrated into host. Several myoblast-mediated gene delivery system had been established, including the gene delivery of human factor IX (hFIX), erythropoietin (EPO) and clony stimulating factor-1 (CSF-1). Results from animal experiments demonstrated that myoblast-mediated gene delivery could be used as gene therapy for some inherited diseases. And recently, some authors have shown great interest in the interaction between myoblast and type I collagen gels. It was found that myoblast could keep on proliferating and differentiating in collagen gels and could form discoid, tubular materials. CONCLUSION Myoblast has great importance in gene therapy and tissue engineering. It is suggested that more efforts should be made in this field.
Objective To study the effect of recombinant adeno-associated virus (rAAV) vector co-expressing human vascular endothel ial growth factor 165 (hVEGF165) and human bone morphogenetic protein 7 (hBMP-7) genes on bone regeneration and angiopoiesis in vivo so as to provide a theoretical basis for the gene therapy of avascular necrosis of thefemoral head (ANFH). Methods Twenty-four male adult New Zealand rabbits were made the ischemic hind l imb model and divided into 4 groups (n=6). The 3rd generation rabbit bone marrow mesenchymal stem cells (BMSCs) were transfected with the following 4 virus and were administered intramuscularly into the ischemic thigh muscle of 4 groups, respectively: rAAVhVEGF165- internal ribosome entry site (IRES)-hBMP-7 (group A), rAAV-hVEGF165-green fluorescent protein (GFP) (group B), rAAV-hBMP-7-GFP (group C), and rAAV-IRES-GFP (group D). At 8 weeks after injection, the blood flow of anterior tibial artery in the rabbit hind l imb was detected by ultrasonographic image. Immunohistochemical staining for CD34 was performed to identify the prol iferation of capillary. Another 24 male adult New Zealand rabbits were made the femur muscle pouch model and divided into 4 groups (n=6). The above 4 BMSCs transfected with rAAV were administered intramuscularly into the muscle pouch. At 8 weeks after injection, X-ray radiography was used to assess orthotopic bone formation, and von Kossa staining to show mineral ization. Results No symptoms of local or systemic toxicity were observed after rAAV injection. At 8 weeks after injection, the ratio of ischemic to normal blood flow and the number of capillaries in group A were the highest among 4 groups (P lt; 0.05). The ratio of ischemic to normal blood flow and the number of capillaries in group B were significantly higher than those in group C and group D (P lt; 0.05). However, there was no significant difference between group C and group D (P gt; 0.05). At 8 weeks after injection, orthotopic ossification and mineral ization were evidently detected in group A and group C, and group A was ber than group C. No obvious evidence of orthotopic ossification and mineral ization were observed in group B and group D. Conclusion rAAV-hVEGF165-IRES-hBMP-7 vector has the biological activities of inductive bone regeneration and angiopoiesis in vivo.
Objective To investigate the inhibitive effect of E2F decoy oligodeoxynucleotides (E2F decoy ODNs) on cultured human retinal pigment epithelial (HRPE) cells.Methods E2F decoy ODNs or scramble decoy ODNs at varied concentrations were put into the HRPE cells mediated by lipofectamineTM2000. The proliferative activity of HRPE was detected by methythiazolyl-terazollium assay, and the competitive combinative activity of E2F decoy ODNs and transcription factor E2F was detected by electrophoresis mobility-shift assay. Results The proliferation of HRPE was inhibited markedly by E2F decoy ODNs at the concentration of 0.2 μmol/L (P=0.002) in a dose-dependent manner but not by scrambled decoy. The results of electrophoresis mobility-shift assay showed that the combinative activity of transcription factor E2F was abolished completely by E2F decoy ODNs. Conclusions E2F decoy ODNs may sequence-specifically inhibit the combinative activity of transcripti on factor E2F,and inhibit the proliferation of HRPE cells.(Chin J Ocul Fundus Dis，2004，20：182-185)
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.
【Abstract】ObjectiveTo construct a recombinant eukaryotic expression vector for human endostatin in order to study the inhibitory effect of liposome-mediated endostatin gene on the growth of human liver carcinoma in nude mice. MethodsHuman endostatin cDNA including IL-2 secreting peptide was cloned into eukaryotic expression plasmid pcDNA3.0 to construct recombinant plasmid pCD-sEndo. pCD-sEndo plasmid was transferred into hepatocarcinoma cell line SMMC-7721 mediated by liposome Dosper, the expression and secretion of endostatin gene was detected by RTPCR and Western blot analysis. The suspension of SMMC-7721 cells was injected subcutaneously at the back of 32 nude mice to establish the model of human liver carcinoma. The mice were divided into 4 groups randomly, and injected with Dosper+pCD-sEndo, Dosper+pcDNA3.0, Dosper and physiological brine separately. Tumor volume was measured by stages. The mice were executed after the drug had been given for 1 week, then the microvessel density (MVD) of the tumor tissue was detected with immunohistochemical method and apoptotic index of tumor cells was measured by TUNEL-stain. ResultsThe eukaryotic expression vector pCD-sEndo was successfully constructed and was confirmed by enzyme digestion and sequence analysis. Expression of endostatin gene was detected in transfected SMMS-7721 cells by RTPCR in vitro, and endostatin protein was also detected in the supernatant of transfected SMMS-7721 cells by Western blot. In vivo study, the growth of human liver carcinoma was inhibited in the group injected with endostatin gene: the average volume of tumor in this group was significantly smaller than that in other groups （P＜0.05）; the average MVD in this group was 6.2±2.5, significantly less than that in the group injected with physiological brine (32.8±6.4), Dosper （27.8±6.4）, or Dosper+pcDNA3.0 (25.5±5.5), P＜0.05. The average apoptotic index of tumor cells in treatment group, brine group, Dosper group and Desper+pcDNA3.0 group was 24.5±7.3, 7.6±2.5, 9.5±3.0 and 11.2±3.6 respectively, it was evidently higher in the treatment group than in the latter three groups. ConclusionHuman endostatin mediated by cation liposome could decrease the microvessel number of implanted human hepatocarcinoma in nude mice. It could also accelerate apoptosis of tumor cells and inhibit growth of tumor.
ObjectiveTo discuss the feasibility of treating noumenon tumor by antiangiogenesis.MethodsRelated literatures of recent 5 years was reviewed.ResultsTumor angiogenesis were related closely with growth, development, metastasis and prognosis of noumenon tumor. It was possible to inhibit the growth and metastasis of noumenon tumor with antiangiogenesis in vitro and vivo.ConclusionAntiangiogenesis will be a new therapy of treating noumenon tumor.
Objective To investigate the expression of multigenes mediated by adenovirus in liver cancer cells and the effects on growth of cells transducted with multigenes. MethodsBy construction of recombinant adenovirus containing human p53, B7-1, GM-CSF, and IL-2 genes (Ad-multigenes), the expression level of target genes in three human hepatocellular carcinoma cell lines and a human hepatocellular cell line L02 was detected using ELISA, immunohistochemistry and FACS assay and the change of growth of these cells and the tumor cell apoptosis were observed. Results The human hepatic cells and liver cancer cells were all sensitive to adenovirus infection. At a MOI of 50 PFU/cell, among the cells examined nearly 90% were positive expression and except IL-2, other three genes were expressed with high efficiency. The growth of Ad-multigenes-transduced liver cancer cell lines was inhibited and apoptosis was induced, but the growth of Ad-multigenes-transduced normal hepatic cell line L02 did not change. Conclusion These results indicate that the adenovirus is an efficient vector for gene transfer into human liver cancer cells. These liver cancer cell lines transduced with multigenes constructed on one recombinant adenoviral vector can highly express target genes and their growth was inhibited, and apoptosis appeared.