Objective To observe the permeability and stability of the transfection of antisense oligonucleotide (ASODN) hybridized epidermal growth factor receptor (EGFR) to retinal glial cells (RG).Methods Phosphorothioate and unmodified EGFR ASODN conjugated with 5′-isothioc yanate (5′-FITC) were encapsulated with or without lipofectin, and then added into human retinal glial cells culture media. The cellular permeability and stability of the transfection were observed by fluorescence microscopy in fixed cells.Results In the absence of lipofectin, phosphorothioate and unmodified EGFR ASODN were found in a few RG cells at 30 minutes, and in about 50% RG cells at 4 hours. Phosphorothioate EGFR ASODN were kept in RG cells for 3-4 hours and disappeared at about 8 hours. In the presence of lipofectin, phosphoro thioate and unmodified EGFR ASODN were found in a few RG cells at 15 minutes and about 70%-80% RG cells at 4 hours. Phosphorothioate EGFR ASODN were kept in cells for 10-12 hours, and phosphorothioate and unmodified EGFR ASODN were disapp eared at about 14 hours and 4 hours respectively.Conclusion 5′-FITC EGFR ASODN encapsulated with lipofectin could enter RG cells and express stably in RG cells. (Chin J Ocul Fundus Dis，2003，19：52-54)
Objective To observe the different effect such as high concentration of glucose and high concentration of insulin on GLUT1 of Rabbit Retinal Muuml;ller Cell in vitro. Methods Rabbit retinal Muuml;ller cells were cultured in vitro with suspended constitution，which were divided as the following groups: common control group，high glucose group，insulin group，high glucose combined insulin group. Laser confocal microscope combined with immunocytochemical and fluorescence staining method to quantitatively analyze the expression condition of GLUT1. Results The expression of GLUT1 has been enhanced obviously by high glucose and high insulin，which locates mainly in the cytoplasm that near to the nucleus. Conclusion Rabbit retinal Muuml;ller cells can express GLUT1，and the expression of GLUT1 can be reinforced by high glucose and high insulin. （Chin J Ocul Fundus Dis，2008，24：265-267）
Objective To observe the dynamic expression of nestin and glial fibrilary acidic protein (GFAP) in the development of retina in rats.Methods In 48 Wistar rats, 24 were divided into 8 groups with 3 rats in each according to their age (1 day, 1 week, and 2, 3, 4, 7, 12, and 20 weeks old). The sagittal freezing sections of the eye were made; nestin/glutamine synthetase (GS) and GFAP/GS were stained by immunofluorescence and were observed under the confocal microscope. Total RNA was extracted from 18 rats which were divided into 6 groups according to the age (1 day, 1 week, and 2, 3, 4, and 12 weeks old) with 3 rats in each. The expression of nestin, GAFA and GS mRNA were detected by realtime quantitative reverse transcription polymerase chain reaction (RT-PCR). Müller cells were cultured from postnatal day 7-12 rats; the expression of nestin and GFAP was detected by immunostaining study. Double immunofluorescence was carried out between nestin/GS and GFAP/GS.Results One day after the birth, nestin positive cells were found in the whole retinal neuroblast layers with elongated retinal progenitor cells; the GFAP positive astrocytes were observed in the inner retina. One week after the birth, Müller glial cells expressed GS and nestin but not GFAP; GFAP positive cells localized in the inner retina.Two to 12 weeks after the birth, the expression of nestin in Müller cells decreased and even disappeared; the expression of GFAP in astrocytes didn't change much. The Müller cells expressed nestin but no GFAP in vitro. The expression of nestin and GFAP mRNA in retina was accordant with the results of immunofluorescence staining.Conclusion In the developing retina, the expression of nestin in Müller cells decreases gradually, and no expression of nestin can be found in adult rats; the expression of GFAP can't be observed in Müller cells in neonatal and adult rats.
【摘要】 目的 探讨成人幕上低级别胶质瘤（WHO Ⅱ级）患者术后生活质量的影响因素。 方法 回顾性分析2008年10月—2010年5月经手术切除病变、术后病理证实为低级别胶质瘤的115例患者临床资料，术后随访6~24个月。以患者年龄、性别、主要临床症状、病变部位、病变大小及病理结果为自变量，以术后Karnofsky评分（KPS）改善为因变量，采用Logistic回归分析研究相关影响因素。采用秩和检验比较不同组间KPS差异。 结果 随访至术后6个月，患者年龄、病变大小、病变部位、切除范围以及是否有癫痫史在KPS比较中，其结果有统计学意义（Plt;0.05）。随访至术后12个月，切除范围和癫痫史对KPS评分已无影响（Pgt;0.05）。病理类型、术前是否存在神经功能障碍与术后KPS改善在单因素和多因素比较中无统计学意义。 结论 患者年龄≤50岁、术前有癫痫史、肿瘤直径≤4 cm、病变表浅、肿瘤全切除的患者术后KPS改善好于年龄gt;50岁、术前无癫痫史、肿瘤直径gt;4 cm、病变深在、肿瘤次全切除的患者。患者术前是否存在神经功能障碍和病理类型与术后生活质量是否改善无明显关系。复发也是影响患者术后KPS改善的因素。【Abstract】 Objective To assess the quality of life in adults with surgically managed cerebral supratentorial low grade glioma (WHO grade Ⅱ) and the relevant factors. Methods We retrospectively analyzed the clinical data of 115 patients with histologically proven supratentorial low grade glioma enrolled at West China Hosptial from October 2008 to May 2010. Follow-up lasted for 6 to 24 months after operation. Logisitc regression analysis is used to test the relevant factors with age, gender, main clinical manifestations, lesion location, lesion size and pathological results as the independent variables, and Kamofsky postoperative scale (KPS) scores as dependent variable. KPS scores of different groups were analyzed using the rank test. Results After 6 months of follow-up, we found that age, size, location, extent of surgical excision and eplispy history showed a statistical significance in KPS comparison (Plt;0.05). Till the 12th month in the follow-up, the extent of surgical excision and eplispy history were not statistically significant any more (Pgt;0.05). Histology type and neurological deficit had no relationship with KPS improvement in both single factor analysis and multivariate analysis. Conclusions Patients with an age older than 50 years, preoperative epilepsy history, the largest diameter of the tumor less than 4 cm, shallow lesions, and complete tumor resection have a better KPS improvement after operation than those with corresponding opposite conditions. There is no obvious relationship between histology type, neurologic deficits and KPS improvement after operation. Recurrence is also a factor influencing KPS improvement after operation.
The human hereditary retinal degeneration is one of the main cause of irreversible blindness in the world. the mechanisms leading to retinal photoreceptor degeneration are not entirely clear. However, microglia acting as innate immune monitors are found to be activated early in retinal degeneration in many retinitis pigmentosa animal models. These activated microglia are involved in phagocyte rod cell fragments of degenerated retina, and also produce high levels of cytotoxic substances such as pro-inflammatory cytokines and chemokines, which aggravate the death of adjacent healthy photoreceptor cells. It suggests that microglia activation plays an important role in photoreceptor degeneration. At the same time, a series of studies have confirmed that some drugs can prevent or reduce neuronal death and slow the occurrence and progression of retinal degeneration by interfering with abnormal activation of microglia. It is expected to be a new choice for the treatment of hereditary retinal degeneration.
ObjectiveTo observe the effect of conditional knocking out (KO) vascular endothelial growth factor (VEGF) gene on the mouse model of oxygen induced retinopathy (OIR).MethodsThe conditional VEGF KO mice were generated using Cre-Loxp technology, resulting in the deletion of VEGF in a portion of Müller cells permanently in mouse retina. Cre positive was CKO mice, Cre negative was NKO mice. OIR was induced by keeping mice in 75% oxygen at postnatal 7 days (P7) to P12 and in room air from P12 to P17 (each 20 mice for CKO and NKO, respectively). The mice mortality was analyzed. At day P17, the percentage of retinal avascular area was calculated using retinal flat-mounting with fluorescence angiography, the number of vascular endothelial cell nucleus breaking through retinal inner limiting membrane was counted with hematoxylin eosin (HE) staining of retinal sections, and the expression of hypoxia-inducible factor-1α (HIF-1α) was detected by immunofluorescence analysis. ResultsDuring the development of OIR, the mortality rate of CKO mice (65.00%) was higher than that of NKO mice (30.00%) with the significant difference (x2=4.912, P=0.027). At day P17, all the mice retinas were harvested. The retinal fluorescence angiography displayed that the normal retinal vascularization of CKO mice was delayed, and large avascular areas were observed. Meanwhile, rare new vascular plexus was found in CKO mice and the thickness of whole retina decreased dramatically. In contrast, NKO mice developed larger area of normal retinal vascular network structure with higher blood vessel density and more new vascular plexus with obvious fluorescein leakage. The percentage of avascular area in CKO mice [(28.31±11.15)%] was higher than NKO mice [(16.82±7.23)%] with the significant difference (t=2.734, P=0.014). The HE staining of retinal sections indicated smaller counts of vascular endothelial cell nucleus breaking through retinal inner limiting membrane in CKO mice (26.10±6.37) when compared to NKO mice (28.80±7.59) , the difference was significant (t=2.437, P=0.016). The immunofluorescence analysis showed stronger expression of HIF-1α in CKO mice than NKO mice, which was mainly located in the retinal ganglion cell layer.ConclusionsThe local VEGF gene knockout partially inhibits retinal neovascularization in OIR mice. However, it also suppresses the normal retinal blood vascular development with a decrease of OIR mice survival ability.
Objective To observe the relationship between retinal microglial activations and ganglion cell (RGC) damages in early-stage diabetic rats. Methods A total of 20 SpragueDawley（SD）rats were randomly divided into 4 groups (each with 5 rats): 1 month control group, 1 month diabetes group, 3 month control group, 3 month diabetes group. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ). The RGCs of all rats were retrograde labeled by carbocyanine dye DiI injected at the superior colliculi.Microglial cells and RGCs in retinal flat-mounts and sections were stained immunohistochemically and recorded under confocal microscope.Results The diabetic microglial cells were amoeboid and ovoid with fewer processes on retinal flat mounts. The density of microglial cells which phagocytosed DiI particles in the RGC layer significantly increased in the 3month diabetes group（P＜0.01）. The density of microglial cells in the RGC layer significantly increased in the 1- and 3- month diabetes group（P＜0.05）. However there were more microglial cells in the RGC layer in the 3- month diabetes group than the 1-month diabetes group（P＜0.0001）. Significant correlation was found between the amount of microglial cells and that of RGCs in the early-stage of diabetes. Conclusions Microglial cell activation has close relationship with the RGC damages in early-stage diabetic rats.
Objective:To study combination effects of gamma;-ray radiation and hyperthermia on the in vitro cell proliferation of cultured human retianl glial cells in order to explore possible application of the combination treatment for proliferative vitreoretinopathy. Methods:Cultured human retinal glial cells were tread by radiation, hyperthermia,or a combination of the two.Cell proliferation was evaluated by MTT method. Results:gamma;-ary irradiation of 100cGy or 300cGy was not effective in suppressing proliferation of the retinal glial cells,neither was the heat treatment at 42℃ or 43℃ for 30 min.Howver,combination of hyperthermia at 42℃ for 30min with 300cGy irradiation suppressed cellular growth of the retinal glial cells to 25.2% of the control.Combination treatment of 43℃，30 min hyperthermia and 300cGy irradiation was more effective. Conclusion:A combination of low dose radiation and mild hyperthermia is effective in the suppression of frowth of cultured human glial cells,and the effects were found to be synergistic.It is expected that the synergistic effects will lower the radiation dose and and also reduce the possible side effects of radiation in the treatment of proliferative vitroretinopathy. (Chin J Ocul Fundus Dis,1998,14:29-32)