Epigenetic modifications such as DNA methylation, histone post-translational modifications, non-coding RNA are reversible, heritable alterations which are induced by environmental stimuli. Major risk factors of diabetes and diabetic complications including hyperglycemia, oxidative stress and advanced glycation end products, can lead to abnormal epigenetic modifications in retinal vascular endothelial cells and retinal pigment epithelium cells. Epigenetic mechanisms are involved in the pathogenesis of macular edema and neovascularization of diabetic retinopathy (DR), as well as diabetic metabolic memory. The heritable nature of epigenetic marks also playsakey role in familial diabetes mellitus. Further elucidation of epigenetic mechanisms in DR can open the way for the discovery of novel therapeutic targets to prevent DR progression.
Thirteen patients with intractable nonunions of fractures of long bones were sucessfully treated by a combination of internal fixation and implantation of bBMP. There was an average of 1.5 operative procedures per patient in an attempting to establish reunion prior to bBMP implantation. Union was obtained in 12 of the 13 patients exapt in one who gained success from establish the second attempt. The average time requited to union was 4.7 months. No complication was seen.
ObjectiveTo review the research progress of pathogenesis and genetics of alcohol-induced osteonecrosis of the femoral head (AIONFH). MethodsThe relevant domestic and foreign literature in recent years was extensively reviewed. The pathogenesis, the relationship between gene polymorphism and susceptibility, the related factors of disease progression, and the potential therapeutic targets of AIONFH were summarized. ResultsAIONFH is a refractory orthopedic disease caused by excessive drinking, seriously affecting the daily life of patients due to its high disability rate. The pathogenesis of AIONFH includes lipid metabolism disorder, endothelial dysfunction, bone homeostasis imbalance, and et al. Gene polymorphism and non-coding RNA are also involved. The hematological and molecular changes involved in AIONFH may be used as early diagnostic markers and potential therapeutic targets of the disease. ConclusionThe pathogenesis of AIONFH has not been fully elucidated. Research based on genetics, including gene polymorphism and non-coding RNA, combined with next-generation sequencing technology, may provide directions for future research on the mechanism and discovery of potential therapeutic targets.
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.
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 construct a co-expressing vector of human bone morphogenetic protein 2 (BMP-2) and osteoprotegerin (OPG) and to determine the expression of BMP-2 and OPG in myoblast C2C12. METHODS: Using the isolated total RNA from osteosacoma cell line MG63 as a template, the cDNA encoding region of human OPG was amplified by reverse transcription-polymerase chain reaction (RT-PCT) method and cloned into sites EcoR 1 and BamH I of mammalian expressing vector pIRES2-EGFP, and the cDNA encoding region of human BMP-2 was cloned into endonucleases site BstX I. Then the recombinant plasmid pIRES2-BMP-2-OPG was transformed into C2C12 cell line, the expression of OPG and BMP-2 were determined by Western blot assay. RESULTS: The sequence of OPG cDNA obtained was the same as that reported, recombinant plasmid pIRES2-BMP-2-OPG was constructed successfully. Human OPG and BMP-2 co-expression cell line C2C12 was selected and confirmed by Western blot analysis. CONCLUSION: The co-expressing vector of OPG and BMP-2 is constructed and can expressed stably in myoblast C2C12. The co-expression of human OPG and BMP-2 may be logical approach for treatment of osteoporosis and bone metastasis.
Objective To evaluate the host immune reaction against adenovirus mediated human bone morphogenetic protein 2 (Adv-hBMP-2) gene therapy in repairof tibial defects. Methods Twelve goats were made 2.1 cm segmental defects in he tibial diaphysis and divided into 2 groups. AdvhBMP2 transfected marrow mesenchymal stem cells(MSCs) and untransfected MSCs were implanted into the defect sites of transfected group(n=7) and untransfected group (n=5), respectively. The defect repair was observed by X-ray films after 4, 8, 16 and 24 weeks of transplantation and cellular and humoral immune reactions to adenovirus were assayed before implantation and after implantation. Results More bony callus was found in the bone defects of transfected group. The healing rates were 6/7 in transfected group and 2/5 in untransfected group, respectively at 24 weeks after implantation. The mixed culture of lymphocytes and MSCs showed that the lymphocytes stimulation indexes (SI) increased 14 days after implantation, and there was significant difference between the transfected group (4.213±1.278) and the untransfected group(－0.310±0.147，Plt;0.05); SI decreased after 28 days, but there was no significant difference between the transfected group (2.544±0.957) and the untransfected group (3.104±0.644，Pgt;0.05). After 14, 28, 49, and 120 days of treatment, the titer values of neutralizing antibody against Adv-hBMP-2 (log0.1) were 2.359±0226, 2.297±0.200, 2.214±0.215 and 2.297±0.210 in transfected group, and -0.175±0.335, -0.419±0.171, 0±0.171 and 0.874±0.524 in untransfected group, being significant differences betweentwo groups(Plt;0.05). Conclusion Adenovirus mediated BMP-2gene therapy can cause cellular and humoral immune reactions against adenovirus, which can eliminate the influence of adenoviral genes and proteins within a certain period.
Objective To observe the mutation frequency and the characteristics of rentinitis pigmentosa (RP)1 gene in the Chinese patients with autosomal dominant (AD) RP or sporadic RP (SRP), and to evaluate their potential effects on the pathogenesis of RP. Methods Fifty-five members from 7 Chinese families with ADRP, 30 patients with SRP, and 75 healthy adults were recruited. Polymerase chain reaction (PCR) and direct DNA sequencing were used to detect the sequence mutation in the entire coding region and splice sites of RP1 gene. Univariate analysis and multivariate analysis were used to detect the effect of RP1 gene mutation sites on RP. Results Four coding sequence variants were detected in the codes of 852，872，921 and 939 at the exon 4 of RP1 gene. The R872H alteration, which was found in both ADRP families and patients with SRP, showed positive correlation with RP confirmed by the multivariate logistic regression analysis. The P903L alteration was only found in ADRP families but not in the patients with SRP or the healthy adults. Conclusions The R872H alteration in the RP1 gene is likely to increase the risk of RP, and may be a susceptible gene of RP. Whether the P903L alteration is a diseasecausing factor needs to be further studied.
Objective To investigate the disease-causing gene of Stargardt disease. Method Fifteen patients with Stargardt disease were analyzed with 11 primers of the 11 exons of ABCR gene by using PCR-SSCP and DNA direct sequencing techniques. Results Three newly detected disease-causing mutations were found. Among those mutations, one is a frameshift mutation and others are single base transition. Conclusion This research confirmed that ABCR gene is associated with Stargardt disease, and 3 new mutations of ABCR gene were found. (Chin J Ocul Fundus Dis,2000,16:240-243)
X-linked retinoschisis (XLRS) is a rare X-linked inherited retinal disorder, caused by mutations in retinoschisin 1 (RS1) gene. Three XLRS mice were established, providing ideal systems to study the mechanism and treatment methods for XLRS. RS1 gene mutations can induce abnormal secretion or adhesion function of RS1 protein. In the past year, phase I clinical trials for XLRS has begun in USA, using adeno associated virus (AAV, AAV8 or AAV2)-mediated gene delivery. With the rapid development of new generation of AAV vector that can transduce more retinal cells through intravitreous delivery, gene therapy for XLRS will have a brighter future.