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 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 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.
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 study how CD73 is shed from the retinal pigment epithelium (RPE) surface.MethodsCD73 shedding was induced by treating RPE with lipopolysaccharides (LPS) and TNF-α. After Phospholipase C (PLC) or pan matrix metalloproteinase (MMP) inhibitors were added, surface amount of CD73 was evaluated by flow cytometry (FACS). Then selective inhibitors or their corresponding siRNAs of MMP-2 and MMP-9 were applied to the treatments of RPE; and their effects on induced CD73 shedding were evaluated by FACS. By site directed mutagenesis, mutations were introduced to Lys547-Phe548 coding sites of CD73 cDNA, which was cloned in a pcDNA mammalian expression vector. Both wt-CD73 and mutated-CD73 were over expressed in CD73-/- RPE and their induced shedding was compared.ResultsLPS and TNF-α induced CD73 shedding from RPE was completely blocked by the addition of pan MMP inhibitor but not PLC inhibitor. Selective MPP-9, but not MMP-2, inhibitor or its siRNA blocked CD73 shedding. In CD73-/- RPE induced CD73 shedding was happened to overexpressed wt-CD73 but not Lys547-Phe548 sites mutant CD73.ConclusionMMP-9 is responsible for shedding CD73 from RPE through hydrolyzing its Lys547 -Phe548 sites.
Epigenetics refers to the changes in gene expression level and function caused by non-genetic sequence changes. It can provide the time, location and mode of the genetic information for the execution of DNA sequences, including DNA methylation, histone modification, non-coding RNA and chromatin remodeling. Studies had shown that epigenetics plays an important role in the development of diabetic retinopathy (DR), and it had been found that epigenetic-related treatment regimens had a certain effect on the treatment of DR through animal experiments and in vitro experiments. It was benefit to regulate the development of diabetes and its complications by depth study of DNA methylation, histone modification, miRNA and metabolic memory. An understanding of changes in gene transcriptional mechanisms at the epigenetic level could help us to further study the prevention and control of diabetes and its complications, and to provide new ideas for treatment.
In thiis study,we show thai carbachol stimulates the accumulation of inositol phosphates(InsPs)in human rellnal pigment epithelium (RPE)cells and atropine blocks the carbachol-induced effect ,suggesting the existence of musearinie acelyleholine receptors in human RPE cells. In contrast,noradrenaline,serotonin, cpidermal growth factor (EGF),isoproterenol,and NECA (5'-[N-ethyl]-carboxamido-adenosine)do not influence the basal levels of InsPs.Moreover,isoprmerenol and NECA do not affect the carhaehol elevated levels of InsPs.EGF,howcvcr,does potentiate the carhaehol stimulated elevation of InsPs in a dose-dependent manner ,suggesting an interaction between EGF and musearinie receptors in cultured human RPE cells. （Chin J Ocul Fundus Dis,1994,10:220-222）
ObjectiveTo investigate the difference of DNA methylation before and after bariatric surgery.MethodThe relevant literatures of the research on the changes of DNA methylation level and gene expression regulation in blood and tissues before and after bariatric surgery were retrieved and reviewed.ResultsDNA methylation was an important method of epigenetic regulation in organisms and its role in bariatric surgery had been paid more and more attention in recent years. Existing studies had found that there were changes of DNA methylation in blood and tissues before and after bariatric surgery. The degree of methylation varies with different follow-up time after bariatric surgery and the same gene had different degrees of methylation in different tissues, and some even had the opposite results.ConclusionsDNA methylation levels before and after bariatric surgery are different in different tissues. And studies with larger sample size and longer follow-up time are needed, to further reveal relationship among DNA methylation, obesity, and bariatric surgery.