【作者】 马宁；

【导师】 吴仁毅；

【作者基本信息】 浙江大学 ， 眼科学， 2011， 硕士

【摘要】 缝隙连接(gap junction, GJ)为沟通相邻细胞胞质的胞间通道,其在细胞膜上紧密聚集成簇,通过介导细胞间缝隙连接通讯(gap junctional intercellular communication, GJIC)参与胞间物质、能量的交换和信息的传递,因而对细胞的生长、发育和分化,神经冲动的传导以及内环境稳定的维持具有重要作用。缝隙连接蛋白(Connexins, Cxs)为一类具有较高同源性的蛋白超家族,是构成GJ的基本单位。在眼部,Cxs已被证实存在于角膜、晶状体、虹膜、睫状体、视网膜等多处。Connexin 43作为主要的缝隙连接蛋白,在睫状体中主要表达于双层上皮细胞,而在睫状肌和基质中则未见表达。鉴于Cx43特定的表达位置及其功能上的重要性,越来越多的研究者开始关注其与房水生成之间的联系,并围绕Cx43的磷酸化调控,以及Cx43与房水生成相关机制进行了一些列相关研究。在眼部,一氧化氮合酶(nitric oxide synthase, NOS)含量丰富,其催化生成的一氧化氮(nitric oxide, NO)作为一种活跃的信号分子,可以通过激活腺苷酸环化酶(adenylate cyclase, AC)与鸟苷酸环化酶(guanylyl cyclase, GC)来增强细胞内第二信使环磷酸腺苷(adenosine 3’,5’-cyclic monophosphate)和环磷酸鸟苷(guanosine 3’,5’-cyclic monophosphate, cGMP)的表达。后者作为细胞间重要的第二信使在调控Cx43表达和通道功能上起重要作用,且一些药物能够加强或者减弱该作用。眼内可能存在多种信号通路,通过调节Cx43的表达和通道功能而影响房水生成过程。本研究旨在研究缝隙连接蛋白Cx43在离体人眼和牛眼睫状体的表达与分布,以及NO对其表达的调控作用,我们目前的研究结果提示NO作为一种有效的刺激物能够促进睫状体双层上皮细胞间Cx43的表达,并且进一步证实了NO的该种作用主要依赖于其下游的cGMP/PKG及cAMP/PKA信号通路的激活。本研究对进一步研究房水生成分子机制,了解某些抗青光眼药物的降眼压机制具有重要意义。具体包括：第一部分一氧化氮合酶和鸟苷酸环化酶在人眼睫状体和小梁网中的表达及分布的研究[研究目的]研究神经元型一氧化氮合酶(neuronal nitric oxide synthase, nNOS),诱导型一氧化氮合酶(inducible nitric oxide synthase, iNOS),内皮型一氧化氮合酶(endothelial nitric oxide synthase, eNOS)及鸟苷酸环化酶(guanylyl cyclase, GC)在人眼睫状体和小梁网的表达与分布。[材料和方法]收集10例眼外伤后48小时内摘除的病人眼球(排除其他眼部病史),常规石蜡包埋、切片,HE染色光镜下观察。切片脱色素后以免疫组织化学EnVision二步法检测nNOS、iNOS、eNOS及GC在睫状体中和小梁网中的表达,以磷酸缓冲液(PBS)代替上述各种一抗作为阴性对照。光镜下读片以显色强度和阳性细胞密度相结合作为定性指标。[研究结果]三种一氧化氮合酶(NOS)及鸟苷酸环化酶(GC)在离体人眼睫状体上皮、睫状肌、小梁网和Schlemm’s管内皮中均有广泛表达。在睫状体中,nNOS表达较强,主要表达在色素上皮(PE)和非色素(NPE)上皮细胞胞浆的顶端交界处。iNOS及eNOS在PE与NPE两种上皮细胞胞浆内均匀表达。在部分睫状突中,GC在PE和NPE细胞胞浆中均匀分布,而在其他睫状突中,GC在PE和NPE细胞胞浆顶端交界处表达较强。[研究结论]三种NOS亚型及GC在离体人眼睫状体上皮、睫状肌、小梁网和Schlemm’s管内皮中均有广泛表达。nNOS和GC的特定表达模式提示NO/cGMP信号通路其可能参与了人眼睫状体跨上皮细胞间离子转运,因而可能参与了房水生成的调节过程。第二部分缝隙连接蛋白Connexin43及Connexin40在离体人眼和牛眼睫状体中的表达[研究目的]研究两种常见的缝隙连接蛋白即缝隙连接蛋白43(Connexin43, Cx43和缝隙连接蛋白40(Connexin40, Cx40在正常人眼和牛眼睫状体的表达及分布。[材料和方法]收集10例眼外伤后48小时内摘除的病人眼球(排除其他眼部病史)和10例当地屠宰场新鲜摘取的牛眼球(排除眼球破裂和眼内炎眼球),常规石蜡包埋、切片,HE染色光镜下观察。切片脱色素后以免疫组织化学EnVision二步法检测Cx43和Cx40在离体人眼睫状体中的表达,以及Cx40在离体牛眼睫状体的表达。以磷酸缓冲液(PBS)代替上述各种一抗作为阴性对照。光镜下读片以显色强度和阳性细胞密度相结合作为定性的指标。[研究结果]Cx43在离体人眼及牛眼睫状体中均存在基础表达。其阳性染色密集分布于色素上皮(PE)和非色素(NPE)上皮细胞胞浆.顶端交界处。部分见于PE细胞交界处与其胞浆内。Cx40在离体人眼睫状体组织呈阴性表达。[研究结论]Cx43在离体人眼和牛眼睫状体上皮的分布模式, Cx43通道可能作为人眼和牛眼睫状体上皮细胞间主要的缝隙连接通道来参与房水生成。第三部分NO-cGMP/cAMP信号通路对人眼与牛眼睫状体Connexin43表达影响的研究[研究目的]研究一氧化氮(Nitric Oxide, NO)及其下游信号分子环磷酸腺苷(Adenosine 3’,5’-cyclic monophosphate, cAMP)和环磷酸鸟苷(Guanosine 3’,5’-cyclic monophosphate, cGMP)对离体人眼和牛眼睫状体缝隙连接蛋白43(Connexin43,Cx43)表达的调控作用及可能机制。[材料和方法]蛋白印迹(Western blot)法分别检测离体人眼和牛眼睫状体中Cx43蛋白的表达。将睫状体组织分别与与NO供体硝普钠(SNP,100gM)、cAMP替代物8-Bromo-cAMP (250μM)和cGMP替代物8-Bromo-cGMP (500μM)共同孵育18小时,并用上述药物下游通道的抑制剂阻断其效应,半定量分析对照组、SNP组、8-Bromo-cAMP组、8-Bromo-cGMP组和各抑制剂组Cx43蛋白表达的差异。[研究结果]蛋白印迹法检测到Cx43在离体人眼及牛眼睫状体存在基础表达。SNP,8-Bromo-cAMP,8-Bromo-cGMP能在18小时内显著增加Cx43蛋白的基础表达,PKA抑制剂H89在一定程度上抑制了SNP和8-Bromo-cAMP对Cx43蛋白表达量的上调。PKG抑制剂KT5823在一定程度上能够抑制SNP和8-Bromo-cGMP对Cx43蛋白表达量的上调,sGC抑制剂ODQ在一定程度上可抑制SNP对Cx43蛋白表达量的上调。[研究结论]本研究提示NO能上调人眼和牛眼睫状体Cx43的表达,该调节作用可能由cAMP/PKA和cGMP/PKG两条信号通路所介导。推测NO可能参与了人眼和牛眼房水生成的调节。更多还原

【Abstract】 Gap junctions (GJ) are clusters of intercellular plasma membrane channels that act as conduits for the direct cell-to-cell communication by allowing intercellular exchange of matter, energy and information via gap junctional intercellular communication (GJIC), which is thought to play an important role in the control of variety cellular functions, maintenance of metabolic homeostasis and neuronal transmission.Connexins (Cxs) are a mulitigene family which have the same basic molecular structure to compose gap junctions between adjacent cells. To date, Cxs have been reported with wild distributions in the eye, for instance in cornea, lens, iris, ciliary body and retina, etc. As the major member of Cxs, Cx43 has been proved localized to the PE-NPE interface of ciliary epitheliums to form gap junction but not found in the ciliary muscle in a number of species. Considering its special location and crucial role, more and more studies were focus on Cx43 and begun to investigate on its phosphorylation and relationship to aqueous humor formation.Nitric oxide (NO) which synthesized by the enzyme nitric oxide synthase (NOS) is an important signaling molecule in the vertebrate annimal’s eyes, it can activate the soluble guanylate cyclase (sGC) and adenylate cyclase (AC) to induce the second messenger molecules such as cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) expression, the laters are important regulators in Cx43 channels and some drugs can enhance or reduce their effects. There may exist many signal pathways to influence aqueous humor formation via regulating of Cx43 gap junction.This study aimed to investgate the distribution of Cx43 in human and bovine ciliary body and also the regulation of NO on it. The results indicated that as a effective stimulant, NO can enhance the expression of Cx43 in the two-layers ciliary epithelium, and this effect possiblly depended on the activation of its downstream signal channels such as cGMP and cAMP. Hence our work takes an important role in further studies of aqueous humor generation mechanism and can help to kown more about the action mechanism of some anti-glaucoma medicines,including:PART IExpression of Nitric Oxide Synthase and Guanylate Cyclase in the Human Ciliary Body and Trabecular Meshwork【Purpose】To investigate the expression and distribution of nitric oxide synthase (NOS) isoforms and guanylate cyclase (GC) in human ciliary body and trabecular meshwork.【Methods】Sections of human ocular anterior segments were used. Expression of three NOS isoforms (i.e. neuronal NOS, nNOS; inducible NOS, iNOS and endothelial NOS, eNOS) and GC were assessed by immunohistochemical staining using monoclonal or polyclonal antibody of NOS and GC.【Results】Expression of three NOS isoforms and GC were observed in the ciliary epithelium, ciliary muscle, trabecular meshwork and the endothelium of the Schlemm’s canal. Immunoreactivity of nNOS was detected mainly along the apical cytoplasmic junction of the non-pigmented epithelium (NPE) and pigmented epithelial (PE) cells, while iNOS and eNOS were evenly distributed in the cytoplasm of both the PE and NPE. In some ciliary processes, GC was shown to be evenly distributed in-the cytoplasm of the PE and NPE, while in the others it was expressed mainly along the apical cytoplasmic junction of the PE and NPE. NOS and GC were evenly distributed in the ciliary muscle cells, trabecular meshwork and the endothelium of the Schlemm’s canal.[Conclusions]The distribution patterns of nNOS and GC in human ciliary epithelium suggest the possible involvement of NO and cyclic guanosine monophosphate (cyclic GMP, cGMP) signalling pathway in the intercellular communication between the PE and NPE cells, and may play a role in both processes of aqueous humor formation and drainage.PART IIExpression of Connexin43 and Connexin40 in Human and Bovine Ciliary Bodies [PURPOSE]To investigate the expression of connexin43 (Cx43) and connexin40 (Cx40) in the dual-layered ciliary epithelium of human and bovine eyes. [METHODS]Sections of human and bovine ocular anterior segments were used. Expression of Cx43 and Cx40 were tested using rabbit polyclonal antibody by immunohistochemistry methods. 【RESULTS】Expression of Cx43 was observed in ciliary epithelium of human and bovine but not in the ciliary muclse. Immunoreactivity of it was detected mainly along the apical cytoplasmic portions of pigment ciliary epithelial (PE) cells and the non-pigmented ciliary epithelial (NPE) cells with high concentration. However, there was no staining signal of Cx40 detected in human ciliary body.【CONCLUSIONS】The distribution patterns of Cx43 in human and bovine ciliary epithelium suggest that Cx43 may be a major component of PE-NPE gap junction channel to mediate intercellular communication between PE and NPE cells, and may play a role in the processes of aqueous humor formation.PARTⅢNitric Oxide-Mediated Regulation of Connexin43 Expression in Human and Bovine Cillary Bodies【PURPOSE】To investigate the potential role of Nitric Oxide (NO) and its downstream signaling molecules cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) in the regulation of Connexin43 expression in human and bovine ciliary bodies.【METHODS】The expression level of Cx43 protein in human and bovine ciliary bodies was measured by Western blot, before or after incubated with different drugs. Tissues were exposed for 18 hours to sodium nitroprusside (SNP, an NO donor, 100μM), 8-Bromo-cGMP(an cGMP analogue,500μM),8-Bromo-cAMP(an cAMP analogue, 250μM) respectively. Some experiments were conducted in the presence of ODQ (an soluble guanylate cyclase inhibitor,50μM), KT5823 (an Protein Kinase G inhibitor, 2μM), H89 (an Protein Kinase A inhibitor, lOμM) for 30 minutes before.[RESULTS]Incubation of ciliary bodies with SNP enhanced the basal expression of gap junction protein Cx43.8-bromo-cGMP and 8-bromo-cAMP exerted effects similar to SNP. Moreover, the synergistic action of SNP on Cx43 expression was significantly prevented by OQD, KT5823 and H89. The up-regulation effects of 8-bromo-cGMP and 8-bromo-cAMP were prevented by KT5823 and H89 respectively.[CONCLUSIONS]In short, these results suggested a possible involvement of NO/cGMP/PKG and NO/cAMP/PKA signal pathway in the control of Cx43 expression in human and bovine ciliary bodies. All these findings will open a new window toward our further understanding of the relationship between NO and aqueous humor production.更多还原