Objective To investigate the expression of T cell receptor (TCR) Vβ8.3 gene on CD4+ T lymphocytes in the rats with experimental autoimmune uveoretinitis (EAU). Methods Eighteen Lewis rats were divided into EAU, complete Freund′s adjuvant, and the control group. Inter photoreceptor retinoid-binding protein (IRBP) R16 peptide was synthesized using Fmoc procedure for induction of EAU. Magnetic absorption cell sorting (MACS) me thod was used to isolate the CD4+T lymphocytes from the spleen of the rats. Flow cytometry was used to monitor the efficiency of isolation. The expression of TCR Vβ8.3 gene segment on CD4+T lymphocytes was determined by fluorescent quantitative polymerase chain reaction. Results EAU was successfully induced in the Lewis rats immunized with IRBP R16 peptide. The proportion of CD4+T lymphocytes isolated by means of MACS was statistically higher than that before isolation (P＜0.001). The expression of TCR Vβ8.3 gene segment on CD4+ T lymphocytes in EAU rats was significantly higher than that in the control (P＜0.05). Conclusions There is a predominant usage of antigen-specific TCR Vβ 8.3 gene in EAU rats induced by IR BP R16 peptide, which may serve as a target for immunotherapy of EAU. (Chin J Ocul Fundus Dis，2004，20：165-167)
Objective
To investigate the polymorphism of the vitamin D receptor gene (VDR)TaqⅠin relation to diabetic retinopathy.
Method
Fragment length discrepant allele specific PCR(FLDAS-PCR) were used to determine VDR genetypes in 158 patients with diabetic retinopathy and in 198 normal subjects.
Results
The frequency distribution of VDR genotypes in diabetic retinopathy patients was 106 (67.1%) in TT, 33(20.9%) in Tt, 19(12.0%) in tt; and in normal persons was 165 (83.3%) in TT, 23(11.6%) in Tt, 10 (5.1%) in tt. There was a significant difference between diabetic retinopathy patients and normal persons in distribution of VDR gene TaqⅠgenotypes(Plt;0.05).
Conclusions
There is some distribution alterations of VDR gene polymorphism in diabetic retinopathy patients.
(Chin J Ocul Fundus Dis, 2006, 22: 94-96)
In order to study the expression change of insulin-like growth factor-1 (IGF-1) and its receptor genes in different generations of tendon cell in culture, Dig-labeled synthesized oligonucleotide probes were used to detect the mRNA expression in primary, 6th and 13th generation of tendon cell. The results showed that IGF-1 receptor mRNA was expressed in all of the 3 above generation tendon cells. IGF-1 mRNA was expressed only in primary and 6th generation cells. Tendon cell of 13th generation did not express IGF-1 mRNA. It might suggest that the absence of IGF-1 mRNA expression be one of the causes which led to the decrease of reproductive ability of 13th generation tendon cell.
Purpose
To identify the expression of alternatively spliced mRNA isoforms of the NMDA-R1 in the visual cortex of strabismic cats.
Methods
Two pai rs of normal and strabismic cats were used．The amblyopic cats had been made monocularly esotropic (by tenotomy) at the age of weeks，resulting in behavioral am blyopia．Animals were sacrificed about 6 months by intraperitoneal administration of Nembutal．Cryostat sections of fresh，frozen central visual cortex of the ats were cut to 20 micron thickness．A series of digoxygenin-labelled oligonucle otide probes basing on the human gene sequence were used for ISH．Control probes included sense oligonucleotides and short segment probes which were adjacent to ，but did not，span the splice junctions．A computer-assisted systematic morphometric ounting procedure was used to enumerate hybridising cells．
Results
The number of positive cells expressing NMDA-R1 mRNA in t he strabismic amblyopic cats was decreased，notably in layer IV of visual cortex (P＜0．0001)．The pattern of isoform expression varied between normal and strabismic amblyopic cats with decreased numbers of 1-a，1- b and 1-1 isoforms and apparently increased expression of 1-3 P ＜0．0001)，whereas no significant difference was found for the 1-2 and 1-4 isoforms (P＞0．05)．
Conclusion
Transcriptional inhibition of NMDA-R1 mRNA and of specifie isoforms may underlie the change in receptor expression．Alternatively，preferentialloss of neurones bearing particular NMDA-R1 isoforms and compensation with a proportional increase in cells expressing other isoforms may occurr during the critical period of visual plasticity．
(Chin J Ocul Fundus Dis，2000，16：71-138)
Objective o observe the expression of Notch1 and Delta-like ligand 4 (Dll4) on the fibrovascular membranes in proliferative diabetic retinopathy (PDR), and investigate its relationship with vascular endothelial growth factor receptor 2 (VEGFR2). Methods Fifty-seven PDR patients (60 eyes) who underwent vitrectomy were enrolled in this study. The PDR patients were divided into non-injection group (30 patients, 32 eyes) and injection group (27 patients, 28 eyes). The eyes in injection group received intravitreal injection with ranibizumab at 2 to 7 days before surgery. The preretinal fibrovascular membranes were obtained from the PDR patients during vitrectomy. Eighteen epiretinal membranes were obtained from the non-diabetic patients was served as controls. The real-time polymerase chain reaction (RT-PCR) and immunohistochemical methods were used to detecting the expression of Notch1, Dll4 and VEGFR2. In the meantime, the numbers of the nucleus of vascular endothelial cells in the membranes stained with hematoxylin were counted. Results The immunohistochemical staining revealed that there were positive expression of Notch1, Dll4 and VEGFR2 in all PDR membranes, regardless of the injection of the ranibizumab. The levels of Notch1, Dll4 and VEGFR2 protein in non-injection group were higher than those of injection group (t=3.45, 6.01, 4.08;P=0.030, 0.008, 0.023). In injection group, the number of endothelial cells in the membranes reduced (17.17±2.48) compared with that of the non-injection group (41.50±5.57). There was significant difference in the number of endothelial cells in the membranes between the two groups (t=9.58,P＜0.05). RT-PCR showed that the differences of the mRNA expression of Notch1, Dll4 and VEGFR2 were all statistically significant among the PDR group and control group (H=12.50, 12.50, 12.02;P＜0.05).The expression of Notch1, Dll4 and VEGFR2 in the PDR membranes was higher than that of epiretinal membranes from non-diabetic patients. In the PDR group, the expression of Notch1, Dll4 and VEGFR2 of non-injection group was higher than that of injection group. Spearman correlation analysis showed that the expression of mRNA between VEGFR2 and Dll4 (r=0.83), VEGFR2 and Notch1 (r=0.81), Notch1 and Dll4 (r=0.87) were all significantly correlated (P＜0.05). Conclusions The expression of Notch1 and Dll4 in the PDR membranes are higher than that of the control group, and it is positively correlated with the expression of the VEGFR2. Notch1 and Dll4 play a regulatory rule in the neovascularization in PDR, the acting way may be correlated with VEGFR2.
Objective To investigate the effect of hypoxia on the exp ression and function of integrin receptor αvβ3 of bovine retinal vascular endotheliocytes. Methods Bovine retinal vascular endotheliocy tes in the culture dishes coated by vitronectin was put into the normal and hypoxemic condition, respectively. Enzyme linked immunosorbent assay and cell adhesion analysis were used to detect the expression and function of integrin receptor αvβ3 in bovine retinal vascular endotheliocytes, respectively. Results Under the condition of hypoxia, the expression of αvβ3 increased gradually, and reached the peak at the 48th hour. The expression of αvβ3 at the 60th and 72nd hour in hypoxia group was higher than that in the normal group. Bovine retinal vascular endotheliocytes absorbed more Vn of extra-cellular matrixes (ECM) after cultured under hypoxemic condition for 24 hours.Conclusion Hypoxia may up-regulate the expression of αvβ3, which promote the adsorbability of endotheliocytes.(Chin J Ocul Fundus Dis，2004，20：360-363)
Objective Aseptic loosening of prosthesis is associated with peri prosthetical osteolysis caused by osteoclast activation. Receptor activator of nuclear factor kappa B (NF-κB) l igand (RANKL)/receptor activator of NF-κB (RANK) signalpathway is fundamental in osteoclast activation. To determine whether RANKL antibody can inhibit inflammatory osteolysis in a osteolysis model of mouse. Methods Sixty female BALB/c mice (aged 8-10 weeks, weighing 18-20 g) were selected. The skull bone piece was harvested from 20 mice as the donor of bone graft; the subcutaneous air pouches (2 cm × 2 cm) models were established on the back of the other 40 mice and the skull bone piece was inserted into the air pouches. The 40 mice were equally divided into groups A (negative control group), B (positive control group), C (low-dose RANKL antibody group), and D (high-dose RANKL antibody group). At 1 day after bone graft, 0.5 mL PBS was injected into the pouch of group A, 0.5 mL PBS containing titanium particle into groups B, C, and D. At 2 days before the titanium particle was injected, RANKL antibody (0.1 mL) were injected into the pouch of group C (50 μg/mL) and group D (500 μg/mL), respectively every day for 2 days, and 0.1 mL PBS into groups A and B. At 14 days after bone implantation, the pouchmembranes containing implanted bone were harvested for gross observation and histological analyse. Results All mice survived to the end of experiment, and incisions healed well. The gross observation showed that inflammatory responses, exudation, and vascular proliferation were obvious in group B, and were inconspicuous in groups A, C, and D. The histological analysis showed that significantly more infiltration of inflammatory cells, more obvious bone resorption, more bone collagen loss, and more positive staining area were observed in group B than in groups A, C, and D. There were significant differences in inflammatory cell number, pouch membrane thickness, bone collagen loss, and osteoclast content between group B and groups A, C, and D (P lt; 0.05). Conclusion RANKL antibody can directly blockRANKL/RANK signal pathway, which is an efficient therapy to inhibit bone absorption associated with implant wearing particles.
Objective To investigate the alteration of protein kinase C (PKC) and endothelin system in early diabetic rats, and the effect of specific PKC inhibitor on the expression of retinal endothelin-1 (ET-1). Methods The rats model with streptozotocin(STZ)-induced diabetes were set up. The expression of retinal PKC was detected by enzyme-linked immunoabsorbent assay (ELISA). The expression of retinal ET-1, ET-3, ET-A and ET-B receptor mRNA was determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The alteration of retinal ET-1 mRNA after intravitreal injection of PKC inhibitor GF109203X in diabetic rats was also observed. Results The activities of membranous PKC were significantly increased in 2-week diabetic rats compared with that in normal rats（t=3.296 , P=0.008）, while activities of cytosolic PKC were unchangeable（t=0.138, P=0.894）. The expression of retinal ET-1 mRNA was significantly increased（P=0.008）, while no change was found in expression of ET-3, ET-A and ET-B mRNA（P=0.918，P=0.889，P=0.500）. After intravitreal in jection of 10-5、10-6、10-7 mol/L PKC inhibitor GF109203X in diabetic rats, the expression of retinal ET-1 mRNA was decreased in a dose-dependent manner compared with the control rats. Conclusion Activation of PKC and increased expression of ET-1 could be found in the retina of early diabetic rats, and PKC inhibitor could inhibit the expression of retinal ET-1. (Chin J Ocul Fundus Dis，2004，20：168-171)
Chronic central serous chorioretinopathy (CSC) usually demonstrates frequent recurrence, diffuse leakage and persistent subretinal fluid, which cannot be absorbed, thus lead to photoreceptor damage and poor visual acuity. As glucocorticoids have been implicated in the pathogenesis of chronic CSC, various anti-glucocorticoids oral drugs were used in the clinic to promote retinal fluid absorption and reduce the central retinal thickness of the macula and improve the vision outcomes. In addition, the 5α-reductase-specific inhibitor finasteride, the P450-3A4 inducer rifampicin, circadian rhythmic regulator melatonin, and systemic anti-inflammatory drug methotrexate have also been put into clinical trials for chronic CSC, and achieved certain effects. However, most of the clinical studies on these oral drugs were case reports, but not multi-center randomized clinical trials. The long-term effects of these oral drugs need to be observed and studied further.
ObjectiveTo observe the role of Notch signaling pathway inhibitor in differentiation process of stem cells derived from retinal Müller cells into the ganglion cell.
MethodsRetinas of Sprague Dawley rat at postnatal 10-20 days were dissociated from eye balls. The third passage of Müller cells was used in this experiment, which cultured by repeated incomplete pancreatic enzyme digestion method. The retinal Müller cells were induced in the serum-free dedifferentiation medium. The cell proliferation state was observed under an inverted microscope. The expression of the specific markers Nestin and Ki-67 of retinal stem cells was measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The positive rate of nucleus was detected by Edu. The retinal stem cells was divided into Gamma secretase inhibtor-I (GSI) group and control group, the rate of ganglion cells was counted by using immunofluorescence staining.
ResultsThe cell proliferation had gathered to form a sphere. Immunofluorescence staining showed that the expressions of Nestin and Ki-67 were (92.94±6.48%) and (85.96±6.04%) respectively. Edu positive rate of nucleus was (82.80±6.65)%. RT-PCR and Western blot further confirmed the high expression of Nestin and Ki-67 in the cell spheres but not in the Müller cells. The positive rate of ganglion cells were (16.98±2.87)% and (11.17±0.71)% in GSI group and control group respectively, with the significant difference (t=3.210, P=0.002).
ConclusionNotch signaling pathway is an important regulatory gene in stem cells differentiated into retinal ganglion cell.
