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.
OBJECTIVE: To investigate the expression and distribution of xenoantigen in intervertebral disk of Chinese banna minipig inbred line, and to study the availability of xenograft transplantation of intervertebral disk. METHODS: Samples of intervertebral disk were collected from six Banna pigs of 8 to 11-month-old. The fixation, embedment and slice were performed. α-Gal specific binding lection (BSI-B4) were used as affinity reagents and affinity-immunohistochemistry assays (SABC methods and DAB stain) were conducted to detect the expression and distribution of xenoantigen (α-Gal). RESULTS: alpha-Gal was found in chondrocyte cell and chondrocyte-like cell in intervertebral disk which have the positive yellow-stained particulate aggradation. There was no stain in the matrix, elastic fiber and collagen fiber. CONCLUSIONS: The distribution of xenoantigen is locally in the tissue of intervertebral disk and its expression is weak. This suggests that the intervertebral disk of Banna pig may be alternative donor for xenotransplantation.
OBJECTIVE: To isolate and characterize mesenchymal stem cells (MSCs) derived from bone marrow of Banna minipig inbred line (BMI). METHODS: BMI-MSCs was isolated from bone marrow by density gradient centrifugation and cultured in DMEM (containing 15% bovine serum) at 37 degrees C with humidified 5% CO2. These cultured stem cells were characterized in clonal growth, expression of specific markers and capability of differentiation. RESULTS: Mesenchymal stem cells were proliferative and could be expanded rapidly in vitro. Clonal growth of these cells can be observed when small amount of cells was inoculated. These cells were SH2, SH3, SH4, SB10 and SB21 positive. And it was proved that these cells possess osteo-differentiation ability, up-regulated alkaline phosphatase expression and calcium secretion after osteosupplement was added into the media for several days. CONCLUSION: Mesenchymal stem cells derived from bone marrow of BMI possess the general characters of stem cell.
OBJECTIVE: To observe the heart anatomic and histological structure of the Banna mini-pig inbred-lined and to provide the morphological data for heart xenotransplantation and breeding transgens pig. METHODS: Ten Banna mini-pigs (12-18 months old) were affused and fixed by common coratid artery. The heart were observed and measured by gross anatomy and histology. RESULTS: There were many similarities between the Banna pig heart and the human heart in anatomy and histology. However, the following differences were observed in the Banna pig heart: 1. Azygos vein directly drew into right atrium cordis. 2. The intercalated disk of cardiac muscle was less than that of human. 3. The Purkinje’s fibre was bigger than that of human. CONCLUSION: On the morphology and histology, the structure of Banna pig heart is similar to the heart of human being. It is possible that Banna minipig heart becomes organ donors for xenotransplantation.
OBJECTIVE The major obstacle in pig to human transplantation is acute and hyperacute rejection (HAR) triggered mainly by alpha-galactosyl residues(alpha-Gal) in donor. Since the inbred-line Banna pig(IBNP) and Wuzhishan pig (IWZSP) are highly inbred and may be the potential donor for xenotransplantation, it is important to investigate the reaction between human serum and inbred-line pig tissues as well as the distribution of alpha-Gal in these tissues. METHODS Samples from heart, liver, spleen, lung, kidney, pancreas, small intestine, thymus, skin, lymph node and blood vessels at all levels were collected from four 8 to 11-month-old male IBNPs and one IWZSP. Affinity-immunohistochemistry assays were conducted following routine procedures on paraffin sections with normal human sera of blood type A, B, O, AB and BSI-B4(alpha-Gal specific binding lectin) as the primary antibodies or affinity reagents. Sections digested by alpha-galactosidase were also examined as control. RESULTS Parallel results were obtained from these pig tissues stained against human sera and BSI-B4. There was no significant difference both in the antigens recognized by sera of different blood types or BSI-B4 and in the distribution of alpha-Gal. The best alpha-Gal positive staining was appeared in vascular endothelial cells at all levels and partial parenchyma cells. However, tissues of cartilage, peripheral nerve and muscle were negative. After digested by alpha-Galactosidase, all samples were negative against BSI-B4 and human sera except few positions that showed different staining. CONCLUSION The distribution of target antigen is similar in various tissues of the two kinds of pigs. Though alpha-Gal is the major xenoantigen in IBNP and IWZSP, there may be some unknown antigens related to pig to human transplantation. Possibly the level and distribution of antigen expression in pig tissues are not the first affair to be considered, and these pigs should be genetically modified in order to eliminate rejection in pig to human xenotransplantation.
Choroidal nevus is one of the most common benign melanocytic tumor. The prevalence rate of choroidal nevi is 0.15% - 10.00%, which is high among whites and low among colored people, and is obvious higher in male than that in female. Secondary changes in the surrounding retina of the benign tumor, such as subretinal fluid and choroidal neovascularization, may result in vision loss. This benign tumor carries risks for transformation into malignant melanoma. The factors predictive of transformation into melanoma included greater thickness, subretinal fluid, visual symptoms, orange lipofuscin pigment, tumor location (tumor margin near optic disc), ultrasonography hollowness and absence of halo. Early identification of the related features which impair visual acuity is important for early treatment and better prognosis, and it is especially important to monitor the tendency of malignant transformation. Optical coherence tomography (OCT) could provide detailed information which aid in diagnosing, differentiating and monitoring of choroidal nevi. OCT and optical coherence tomography angiography are emerging as excellent techniques to investigate choroidal melanocytic lesions. The treatment modalities, such as laser photocoagulation, photodynamic therapy and intravitreal anti-vascular endothelium growth factor, have been proved to be effective for choroidal nevi with secondary changes. In the future, the relevant researches should be imposed to provide more detailed information in order to explore the nature and characteristics of this disease.
【摘要】 目的 观察不同种培养基中重组人色素上皮衍生因子(rPEDF)融合蛋白的表达。 方法 将前期研究已构建的pET28aPEDF原核表达重组体转化E.coli BL21大肠杆菌表达宿主菌,酶切鉴定阳性菌落后,分别在M9和LB培养基中用异丙基βD硫代半乳糖(IPTG,IsopropylbetaDthiogalactoside)诱导表达,SDSPAGE电泳检测表达的PEDF蛋白, 美国ImagePro Plus 分析系统进行蛋白定量分析。结果 LB和M9培养基中均获得相对分子质量约54×103的rPEDF融合蛋白。但LB培养基获得的是rPEDF融合蛋白的包涵体,目的蛋白占总蛋白含量为21046%,M9培养基获得的是可溶性的rPEDF的融合蛋白,目的蛋白占总蛋白含量的1231%。结论 不同种培养基中均有rPEDF 融合蛋白的表达。【Abstract】 Objective To observe the express of recombinant pigment epithelial derivative facto (rPEDF) in the different medium. Methods The pET28aPEDF was transformed into E.coli BL21. After the colonies were positive identification which were induced by IsopropylbetaDthiogalactoside in medium M9 and LB. The PEDF protein were detected by SDSPAGE and analyzed by American ImagePro Plus system. Results LB and M9 medium obtained the relative molecular mass about 54×103 rPEDF fusion protein. But LB medium obtained the inclusion bodys of rPEDF fusion protein，the purpose protein account for 21.046%；LB medium obtained the soluble rPEDF fusion protein，the purpose protein account for 12.31%. Conclusion The rPEDF protein was expressed in the different medium.
Objective To detect and analyse the mutations in rhodopsin gene of members in a family affected by autosomal dominant retinitis pigmentosa (ADRP). Methods Using the polymerase chain reaction (PCR), we amplified exon 1-5 of rhodopsin gene in patients with ADRP,and analyzed it with direct sequence measuement. Results The Gly-182-Asp mutation in the rhodopsin gene was detected in most of affected members of this ADRP family, but no mutation was detected in two affected members and the control ones. Conclusion We cannot regard the Gly-182-Asp mutation in the rhodopsin gene as the pathagenic factor of the ADRP family. It is likely there is a new gene next to the rhodopsin gene. (Chin J Ocul Fundus Dis, 2002, 18: 256-258)
ObjectiveTo investigate the impact of L-Phenylalanine on the efficiency of retinal pigment epithelial (RPE) cell derivation from human embryonic stem cells (hESCs) and explore the underlying mechanisms. MethodsH1 hESCs were routinely cultured with mTeSR medium and divided into control and experimental groups. When cells reached over-confluence, spontaneous differentiation was triggered using 10% KSR differentiation medium without bFGF. L-Phenylalanine (0.2 mmol/L) was supplemented in the experimental group from the 3rd week. The expression of RPE markers and Wnt signaling components in the two groups was detected by Real time-RCR, Western blot and Flow cytometry analyses. Purified hESC-RPE cells and PBS were injected into the subretinal space of sodium iodine-induced retinal degeneration rats separately. Retinal function was assessed by ERG 6 weeks after the transplantation. ResultsOn the 7th week, much more pigment cell clumps appeared in the experimental group compared to the control group. Within these areas there were monolayer hexagonal RPE cells full of pigment granules. The experimental group showed significantly higher expression of Pax6, MITF, Tyrosinase, RPE65, Wnt3a, Lef1 and Tcf7 genes than the control group (P < 0.01). Higher expression level of MITF and RPE65 proteins and higher percentage of RPE65 (+) cells (P < 0.01) were detected in the experimental group. 6 weeks after sub-retinal transplantation of hESC-RPE cells, the amplitudes of a-b wave in the transplanted eyes were significantly higher than those in the control eyes (P < 0.01) at the stimulus intensity of 3.0 cd·s/m2. ConclusionsL-Phenylalanine effectively promoted the differentiation of embryonic stem cells into retinal pigment epithelial cells, and its impacts on the Wnt/β-catenin signaling pathway may partially explain the underlying mechanisms. Subretinal transplantation of hESC-RPE remarkably improved the retinal functions of retinal degenerative animal models.
ObjectiveTo observe the expressions of miR-183 and retinal dehydrogenase 11 (RDH11) in exosomes derived from bone marrow mesenchymal stem cells (BMSC), and to preliminarily explore their targeting relationship and their effects on retinal pigment epithelial (RPE) cells. MethodsBMSC from C57BL/6 (C57) mice were isolated and cultured, and BMSC-derived exosomes were identified. BMSC were divided into blank group, simulation blank control group (mimic-NC group), miR-183 simulation group (miR-183-mimic group). C57 mice and retinal degeneration 10 (rd10) mouse RPE cells were cultured with reference to literature methods. RPE cells from rd10 mice were transfected with BMSC exosomes and co-cultured and divided into control group, exosome group, mimic-NC-exosome group (mimic-NC-exo group), miR-183-mimic-exosome group (miR-183-mimic-exo group). The relative expression levels of miR-183, RDH11 mRNA and protein in C57 mice, rd10 mice and RPE cells in each group were detected by real-time quantitative polymerase chain reaction and western blotting. The targeting relationship between miR-183 and RDH11 was analyzed by bioinformatics website and dual luciferase reporter. Cell counting kit 8 was used to detect the effect of miR-183 on BMSC exosomes on RPE cell proliferation; in situ labeling end labeling method was used to detect RPE cells apoptosis. One-way ANOVA was used to compare multiple groups. ResultsCompared with C57 mouse RPE cells, the relative expression of miR-183 in rd10 mouse RPE cells was down-regulated, and the relative expression of RDH11 mRNA was up-regulated, and the differences were statistically significant (t=5.230, 8.548; P=0.006, 0.001). Compared with the blank group and the mimic-NC group, the relative expression of miR-183 mRNA in the exosomes of the miR-183-mimics group was significantly increased (F=60.130, P＜0.05). After 24 h of co-culture, exosomes entered RPE cells. Compared with the mimic-NC-exo group, the relative expression of miR-183 mRNA in RPE cells in the miR-183-mimic-exo group was significantly increased, the proliferation ability was enhanced (t=7.311, P=0.002), and the number of apoptotic cells was decreased (F=10.949, P=0.012), and the differences were statistically significant (t=4.571, P=0.002). Bioinformatics website and dual-luciferase report confirmed that miR-183 has a targeting relationship with RDH11. Compared with the mimic-NC group, the relative expression of RDH11 mRNA and protein in the exosomes of the miR-183-mimic group was decreased, and the difference was statistically significant (t=5.361, 6.591; P=0.006, 0.003). After co-culture, compared with the control group, there was no significant difference in the relative expression of RDH11 mRNA and protein in RPE cells in the exosome group (t=0.169, 1.134; P=0.874, 0.320); The relative expressions of RDH11 mRNA and protein in RPE cells in -183-mimic-exo group were decreased, and the difference was statistically significant (t=5.554, 5.546; P=0.005, 0.005). ConclusionUp-regulation of BMSC-derived exosomal miR-183 promote the proliferation of RPE cells in vitro by targeting the expression of RDH11 and reduce the number of apoptosis.