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Effect of mouse nerve growth factor on interphotoreceptor retinoidbinding protein in the vitreous of diabetic rats at early stages

　　Objective　To observe the effect of intravitreal injection of mouse nerve growth factor (NGF) on interphotoreceptor retinoid-binding protein (IRBP) in the vitreous of diabetic rats at early stages. Methods　Ninety-six male Sprague Dawley (SD) rats were divided into control group (group A, 24 rats) and experimental group (72 rats). The rats in experimental group were induced with streptozotocin injection for diabetic retinopathy model, and then randomly divided into positive control group (group B), normal saline group (group C) and NGF group (group D), 24 rats in each group. The rats in the group A and B were not intervened. The rats were received intravitreal injection with 4mu;l normal saline (group C) or 4 mu;l (0.5 mu;g/mu;l) NGF (group D). At 2, 4, 6 and 8 weeks after injection, IRBP levels were detected by enzymelinked immunosorbent assay (ELISA); hematoxylin-eosin (HE) staining and light microscope were used to observe the morphological changes of the retina; transmission electron microscope was used to observe the retinal ultrastructure.Results　At 2 weeks after injection, there was no significant difference in IRBP expression between group A,B,C and D (F=2.833，P=0.052). At 4, 6, 8 weeks after injection, the differences of IRBP expression between group A, B, C and D were significant (F=22.252, 108.459, 105.726; P=0.000). At different time points after injection, there was no significant difference in IRBP expression of group A (F=1.462, P=0.241), but there were significant differences in IRBP expression of group B, C and D (F=150.98, 63.519, 64.604; P=0.000). Light microscope found that the retinal structure was clear in group A and in group B, C, D at 2, 4 weeks after injection; the retinal thickness were thinner in group B, C, D at 8 weeks after injection. Transmission electron microscope displayed that the structure of rod outer segments was clear in group A and in group B, C, D at 2 weeks after injection; partly unclear structure of rod outer segments and slightly enlarged gap were observed in group B, C, D at 4, 8 weeks after injection. Conclusion　Intravitreal injection with NGF can stabilize the IRBP expression in the vitreous of diabetic rats at early stages effectively.

Value of Superparamagnetic Iron Oxide in the Experimental Autoimmnune Encephalomyelitis Model

【摘要】 目的 在实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)模型中,比较常规T2加权成像(T2weighted imaging,T2WI)、钆二乙三胺五醋酸(gadoliniumdiethylenetriamine pentaacetic acid,GdDTPA)和超顺磁性氧化铁(superparamagnetic iron oxide,SPIO)增强图像之间的差异,探讨巨噬细胞在多发性硬化(multiple sclerosis,MS)炎性活动病灶中的细胞学标志。方法 在EAE模型临床症状的亚临床期、初发期、高峰期,13只复发缓解(relapsingremitting,RR)EAE大鼠模型组和13只正常对照组大鼠在注入对比剂之前均行常规T2WI扫描,接着分别在其尾静脉注入GdDTPA后5 min行T1加权成像(T1weighted imaging,T1WI),再注入SPIO,24 h后行T2WI扫描。扫描完毕后立即处死大鼠取脑,行脑组织切片的ED1免疫组织化学染色和Prussian blue染色。结果 EAE模型组大鼠在第11天出现临床症状(初发期),第14天达到高峰期;MRI检查:SPIO增强图像对EAE病灶的显示较常规T2WI和GdDTPA增强图像好。病理学检查:ED1染色,在SPIO显示为低信号的区域内出现了炎症细胞(以巨噬细胞为主)浸润;Prussian blue染色示病灶内巨噬细胞胞质内出现了蓝染颗粒,沉积部位与T2WI上低信号区对应。对照组大鼠均无异常。结论 SPIO较GdDTPA更好地显示EAE模型中炎性活动性病灶内血管周围以巨噬细胞为主的浸润。

Immunohistological observation on rabbits′retinae after subreinal implantation with inactive chips

Objective　To observe the expression of related proteins of retina after subretinal implantation with inactive chips.Methods　A total of 27 healthy adult New Zealand white rabbits were randomly divided into three groups: operation group (12 rabbits) in which the rabbits were implanted with inactive chips into the interspace beneath retina;shamoperation group (12 rabbits) in which the rabbits were implanted with inactive chips into the interspace beneath retina which was taken out immediately;the control group (3 rabbits).　Animals were sacrified for immunohistological study 7,15,30 and 60 days after surgery.The rabbits in control group group were sacrified for immunohistological study after bred for 30 days.The expressions of glial fibrillary acidic protein (GFAP) and brain derived neurotrophic facor (BDNF) were observed.Results　In operation group, the outer nulear layer of retina thinned, and the cells in the inner nulear layer was disorganized 7,15,and 30 days after the surgery;glial cells proliferated 60 days after surgery; the positive expression of BDNF and GFAP was more than that in the shamoperation and control group.In shamoperation group, the positive expression of BDNF and GFAP was more than that in the control group.No obvious difference of expression of BDNF and GFAP between each time point groups was found.Conclusions　The expression of neroprotective related proteins increased after subretinal implantation with inactive chips suggests that limited neuroprotective effects might be led by the implantation.

敲除与Th2 细胞活化相关的电压依赖钙通道可防止实验性哮喘发生（Knocking down Cav1 calcium channels implicated in Th2 cell activation prevents experimental asthma）

敲除与Th2 细胞活化相关的电压依赖钙通道可防止实验性哮喘发生（Knocking down Cav1 calcium channels implicated in Th2 cell activation prevents experimental asthma） 【摘要翻译】 研究理由: Th2 型细胞参与过敏性哮喘,这些细胞产生的细胞因子( IL-4、IL-5 及IL-13) 在过敏状态时分泌增加。因此, 研究Th2 型细胞表达的对其功能具有重要影响的关键信号分子至关重要。我们既往的研究显示二氢吡¤特异性调控Th2 细胞的功能。目的: 由于二氢吡¤可特异性与活化细胞的电压依赖钙通道( Cav1) 结合并调节其功能, 我们的主要目的是证实Th2 细胞特异性表达功能性的Cav1 相关通道, 抑制其功能可能抑制哮喘。方法: 我们通过定量PCR 和Western blot 检测Th2 和Th1 细胞Cav1 通道表达。我们将Th2 细胞表达的Cav1 的异构体进行测序, 并研究Cav1 反义寡核苷酸( Cav1AS) 是否影响Ca2 + 信号及细胞因子的产生。最后, 我们通过给OVA 鼻腔激发的BALB/c小鼠注射Cav1AS 转染的OVA 特异性Th2 细胞研究Cav1AS在被动免疫哮喘动物模型中的作用, 并通过鼻腔给予此前进行过OVA 及氢氧化铝免疫的BABL/ c 小鼠Cav1AS 和OVA溶液以研究Cav1AS 对主动免疫哮喘模型的影响。检测和主要结果: 我们发现小鼠Th2 细胞而非Th1 细胞表达Cav1. 2和Cav1. 3 通道。转染Cav1AS 抑制了钙通路和细胞因子产生, 并导致Th2 细胞丧失过继Th2 细胞诱导气道炎症功能。鼻腔内给予Cav1AS 可抑制主动免疫导致的哮喘气道炎症和气道高反应性。结论: 这些结果提示Th2 细胞特异性表达Cav1. 2 and Cav1. 3 通道, 以此作为治疗靶点可有效抑制动物模型的哮喘反应。 【述评】 哮喘是一种Th2 型慢性气道炎症反应疾病,目前机制未明。Th2 细胞在此过程中发挥关键作用, 但Th2细胞活化机制不清楚。本文的研究发现Th2 细胞特异性表达Cav1. 2 和Cav1. 3 通道, 以Cav1AS抑制Cav1 通道的表达可抑制Th2 细胞功能进而抑制哮喘炎症反应。该研究揭示了哮喘气道炎症反应的新机制, 为哮喘治疗提供了新靶点。但是, Cav1 通道如何影响Th2 细胞的功能尚需进一步研究。其次, 有些基因在人类和小鼠表达并不一致, 特别是一些异构体的表达水平不同, 甚至在功能上存在很大差异, 因此, 在哮喘患者中Cav1 通道的表达尚待研究。最后, Th2 功能失调在一些自身免疫性疾病中发挥重要作用, 因此, 如证实Cav1 通道可用于人类哮喘治疗, 则该方法可能对其他一些自身免疫性疾病有治疗作用。

ESTABLISHMENT OF A VINCRISTINE-RESISTANT HUMAN RETINOBLASTOMA CELL LINE

PURPOSE:Studying the multidrug resistance(MDR) phenotype occurring in retinoblastoma and its mechanism. METHODS:Using the procedure of stepwise increase in drug concentrations to obtain a retinoblastoma subline which resistant to 600ng/ml vincristine (HXO-RB/VCR). Characteristics of this drug-resistant cell line were investigated by cell counting,drugcontents determinatin,drug sensitivity evaluation and radiation sensitivity test. RESULTS:This cell line was cross-resistant to VDS,MMC VP16，ADM ,DDP,CBP,but not resistant to BCNU and 5-Fu. It was proved to be collaterally sensitive to MTX,and the response to 60Co gamma;-ray was modified slightly in HXO-RB/VCR cell line. Intracellular levels of VCR was much higher in HXO-RB44 cells than in the resistant subline. Those cross-resistances can be reversed by verapamil partly. CONCLUSIONS:MDR and radiation resistance of retinoblastoma can be induced by exposing to VCR and reversed by verapamil partly. （Chin J Ocul Fundus Dis,1997,13: 6-9）

复方血栓通胶囊对糖尿病大鼠视网膜中凋亡相关因子半胱氨酸蛋白酶3、B细胞淋巴瘤/白血病-2及bcl-2相关X蛋白的影响

Effect of diabetic retinopathy on the quantity of endothelial progenitor cells from peripheral blood in rats

　　Objective　To observe the effect of diabetic retinopathy on endothelial progenitor cells (EPCs) from peripheral blood. Methods　Sixty male Wistar rats were divided into control group and diabetes group. The rats in diabetes group were induced with streptozotocin (STZ) injection for diabetic retinopathy model. Flow cytometry was used to identify and count the number of EPCs from peripheral blood at 1 week, 1, 3 and 6 months after injection. All eyeballs were examined by hematoxylin and eosin (HE) staining, periodic acidSchiff's (PAS) staining of trypsin-digested retinal vessels flat preparation and transmission electron microscope. EPCs count, and the relationship between DR morphological changes and EPCs count were compared and analyzed. Results　The quantity of EPCs from peripheral blood at 1 week, 1, 3 and 6 months after STZ injection were 25plusmn;7, 28plusmn;8, 39plusmn;7, 43plusmn;7 cells per 200 000 monocytes respectively, which decreased compared with the control group 45plusmn;4 cells per 200 000 monocytes (F=8.933，Plt;0.01). The quantity of EPCs was gradually increased at 1 week, 1, 3 and 6 months after STZ injection, accompanied with responsive pathological changes of retinal structure and vessels. The thickness of retina at 1 week and 1 month after injection were reduced slightly. The number of retinal ganglion cells reduced, with the time passing by. Endothelial cells were edema, mitochondrial was swollen, capillary basement membrane was thicken, lumen was significant stenosis, lumen occlusion and retinal artery aneurysm were observed at 6 months after STZ injection. Conclusion　The number of EPCs increases gradually throughout the development of DR.

TUMOR NECROSIS FACTOR-α IN EXPERIMENTAL PROLIFERATIVE VITREORETINOPATHY INDUCED BY MACROPHAGES

PURPOSE:To measure the concentration changes of tumor necrosis factor a (TNF-alpha;)in vitreous during the development of experimental PVR induced by macrophages and explore the initial and mediated factors which stimulate the cellular proliferation. METHODS:Rabbit PVR model was induced by macrophages and the vitreous was taken at the 7th,14th,21st and 28th day and 4 eyes in each group. The TNF-alpha; levels in vivreous of the above examinated and control eyes were measured with an ELISA kit. RESULTS:The TNF-alpha; level in the vitreous reached its peak 434mu;g/ml at 21st day in the mod-el, then rappidly decreased to 122mu;g/ml at 28th day. CONCLUSION:The changes of TNF-a in the vitreous of the PVR model were parallel to the natrual phases of the development of PVR,indicating TNF-alpha; may play an important role in initiating and mediating the inflammation and cellular proliferation in the vitreous. （Chin J Ocul Fundus Dis,1997,13: 231-233）

Retinal toxicity of repeated intravitreal injection with bevacizumab in diabetic rats

　　Objective　To observe the retinal toxicity of repeated intravitreal injection with bevacizumab (Avastin) in diabetic rats. Methods　Forty male Sprague Dawley (SD) rats were randomly divided into normal group (Group A, 10 rats) and diabetes mellitus group (30 rats). The rats in diabetes mellitus group were induced with streptozotocin injection for diabetic retinopathy model. And then randomly divided into diabetic retinopathy (DR) group (Group B,10 rats), the rats were not intervened; the left eyes of the other 20 rats were intravitreal injected with bevacizumab 3 mu;l (25 mg/ml) for 3 times as experimental group (Group C) ; the right eyes of the 20 rats were not intervened as experimental control group (Group D). 20 days after last intravitreal injection, retinal function was measured by Flicker Electroretinogram (F-ERG);retinal vascular pattern was determined by fluorescence microscopy of ethidium bromide (EB) stained retinal flat mounts; retinal morphological changes were determined by light microscope on hematoxylin-eosin (HE) stained sections;Thy-1 and VEGF expression was measured by immunohistochemistry staining. Results　F-ERG showed that, the differences of a-and b-waves, the b-wave amplitude and the Ops-wave amplitude in the implicit time between group A, B, C and D were significant (F=33.165, 36.162,19.955, 23.243; P＝0.000); the differences of a-wave amplitude between group A,B,C and D was not significant (F=0.097,P＝0.961). Retinal blood vessel pattern was normal in Group A; retinal vascular vessels were tortuous and irregularly expanded in Group B; retinal vascular vessels of Group C were regular and thinner than Group A; microaneurysm were showed in Group D. Light microscope displayed that the layers of the rat retina of Group A were regular, the retinal architectures of Group B were irregular, the retinal layers were regular in Group C, the retinal layers were irregular in Group D. Immunohistochemistry staining discovered that Thy-1 and VEGF were mainly expressed in ganglion cell layer（GCL）. Conclusion　Repeated intravitreal injection of bevacizumab is toxic to retina of diabetes mellitus rats.

STUDIES OF MONOCLONAL ANTIBODY AGAINST RETINOBLASTOMA CELLS

PURPOSE: To produce monoclonal antibodies directed against tumor-associated antigens expressed of retinoblastoma-derived tissue culture cell line SO-RBS0. METHODS:Hybridization was performed and the specificity of the antibody was tested by immunofluorescent and immunohistochemical methods. RESULTS:Two hybridomas secreted specific monoclonal antibody against retinoblastoma cells were produced ,and the isotype of the monoclonal antibody was IgG2a CONCLUION:The monoclonal antibodies were specific and highly active against retinoblastoma cells and might be used as immunoconjugate.