【作者】 袁洪峰；

【导师】 贺翔鸽；

【作者基本信息】 第三军医大学 ， 眼科学， 2006， 博士

【摘要】 背景及目的:小梁细胞位于前房角的小梁网组织中,含有多种细胞因子受体,能分泌基质金属蛋白酶(MMP)及细胞外基质(ECM)等多种成分,是维持小梁网结构及功能,保持房水外引流通畅的重要因素。小梁细胞功能障碍可导致房水外流阻力增大,眼压升高引发青光眼。研究发现原发性开角型青光眼患者的小梁细胞数量明显低于正常人,小梁网组织中ECM含量增加。因此,深入研究调节小梁细胞增殖、凋亡和分泌功能的分子机制,对明确青光眼的发病机制以及指导青光眼的治疗具有重要的意义。NF-κB是一个广泛存在于细胞中具有多向调节作用的核转录因子,在细胞生长分化、增殖、凋亡、免疫应答、炎症反应等生理及病理过程中发挥重要的调节作用。NF-κB的活化是炎症反应的中心环节,NF-κB的过度活化与许多急慢性炎性相关疾病的发生发展密切相关。在青光眼的发生、发展及临床治疗过程中,时常伴有炎症的发生,尤其是在外伤、葡萄膜炎所致的继发性青光眼的发病过程中,前房炎症反应所产生的大量炎症介质及细胞因子,必然会刺激小梁细胞NF-κB的活化。压力应激反应可导致细胞NF-κB的活化,研究发现原发性青光眼小梁网NF-κB的表达也明显高于正常人,而NF-κB的活化对青光眼的进程将会产生何种影响,目前仍不清楚。NF-κB的活化对细胞生存至关重要,NF-κB可通过直接调控与细胞增殖相关基因及抗凋亡基因的表达,参与细胞增殖与凋亡的调控。此外,NF-κB还可以调控多种基质金属蛋白酶的表达。临床上长期应用糖皮质激素可导致继发性青光眼的发生,而糖皮质激素是NF-κB的拮抗剂,长期应用激素使NF-κB活性被过度抑制,继而引发小梁细胞的凋亡是否是激素性青光眼的发病机制,目前亦尚不清楚。为此,本研究拟以NF-κB为切入点,通过培养人眼小梁细胞,将NF-κB的活性亚基P65/RelA基因转染入小梁细胞,观察NF-κB在小梁细胞中的过度表达对小梁细胞的增殖、凋亡,以及基质金属蛋白酶和TIGR基因转录和表达的影响。另一方面,采用圈套策略,即应用与NF-κB顺式作用元件序列同源的寡核苷酸序列抑制小梁细胞NF-κB的活化,观察其对小梁细胞增殖、凋亡,以及基质金属蛋白酶和TIGR基因的表达变化的影响。本课题试图阐述NF-κB在小梁细胞功能障碍中的作用,了解NF-κB在不同活性情况下对小梁细胞的影响,讨论其与青光眼发生的可能性。材料方法:人眼小梁细胞原代培养及鉴定:利用角膜移植术的供体眼组织,应用组织块培养法培养小梁细胞,采用免疫组化SP法,进行纤维连接蛋白(FN)、层粘连蛋白(LN)、Ⅳ型胶原、TIGR蛋白及Ⅷ因子相关抗原鉴定。细胞分组:采用培养在三到五代的小梁细胞进行实验,随机分为5组,正常对照组(C),空质粒对照组(P65-C),P65质粒组(P65),随机圈套对照组(ODN-C),NF-κB圈套核苷酸组(ODN)P65质粒及NF-κB圈套核苷酸细胞转染:采用脂质体转染方法,利用荧光显微镜及Westem Bolt法检测转染效果。P65质粒转染后小梁细胞培养液TNF-a含量检测:转染后24小时用ELISA法检测细胞培养液中TNF-α的含量。NF-κB对小梁细胞增殖及凋亡的影响:分别应用MTT及~3H-TdR掺入实验检测小梁细胞的增殖情况,用Tunel法检测小梁细胞的凋亡。NF-κB对小梁细胞基质金属蛋白酶(MMP)及其抑制剂(TIMP)表达的影响:应用RT-PCR方法检测小梁细胞MMP-1,MMP-2,MMP-3及TIMP-1,TIMP-2 mRNA的表达情况。NF-κB对小梁细胞TIGR基因表达调控的影响:应用RT-PCR及Western Blot法分别检测TIGR基因mRNA及蛋白质表达。结果:人眼小梁细胞培养成功,免疫组化鉴定LN、FN、Ⅳ型胶原及TIGR基因表达为阳性,Ⅷ因子相关抗原为阴性。P65质粒转染后24小时,小梁细胞P65的表达明显升高,细胞培养上清液中TNF-α含量显著升高。P65高表达抑制小梁细胞的增殖及DNA合成,促进小梁细胞凋亡增加。NF-κB圈套核苷酸同样抑制小梁细胞的增殖及DNA合成,并且促进小梁细胞凋亡的增加。P65高表达促进小梁细胞MMP-1及MMP-3 mRNA的表达,对MMP-2及TIMP-1,TIMP-2的表达无影响。NF-κB圈套核苷酸抑制小梁细胞MMP-1及MMP-3 mRNA的表达,对MMP-2及TIMP-1,TIMP-2的表达无影响。P65高表达抑制小梁细胞TIGR基因mRNA及蛋白质的表达,NF-κB圈套核苷酸促进小梁细胞TIGR基因mRNA及蛋白质的表达。结论:P65促进小梁细胞分泌炎症介质TNF-α,提示NF-κB过度活化可引发小梁细胞出现过度炎症反应。P65在小梁细胞中的过度表达抑制小梁细胞的增殖及DNA合成,并且促进小梁细胞凋亡的发生。提示前房过度炎症反应可能影响小梁细胞的存活,炎症继发性青光眼的发生可能与NF-κB过度活化密切相关。NF-κB圈套核苷酸抑制小梁细胞的增殖及DNA合成,并且促进小梁细胞凋亡的发生,说明NF-κB在正常情况下具有维持小梁细胞数量及功能平衡的作用。提示NF-κB活性过度受抑可能是糖皮质激素性青光眼的发生机制之一。NF-κB活化可促进小梁细胞MMP-1及MMP-3的表达,抑制NF-κB的活化则会抑制小梁细胞MMP-1及MMP-3的表达,说明NF-κB在调控小粱网细胞外基质平衡机制中占有重要的地位。提示长期应用NF-κB抑制剂,可能会导致小梁细胞MMP-1及MMP-3的分泌下降,造成小梁网ECM的过度堆积。NF-κB的活化和抑制可促使小梁细胞TIGR基因表达改变,说明NF-κB对青光眼相关基因TIGR的表达具有反相调控作用。提示NF-κB功能受抑可能是激素性青光眼小梁网TIGR蛋白表达增高的原因之一。更多还原

【Abstract】 Background and Objective:Trabecular meshwork(TM) cells,located in the TM tissues of the chamber angle of eye,contain multiple cytokines and could secret various substances,such as matrix metalloproteinases(MMPs) and extracellular matrix(ECM) components.It plays an important role in maintaining TM structure and function,and facilitating aqueous outflow. Dysfunctions of TM cells would increase aqueous outflow resistance and lead to elevation of intraocular pressure and development of glaucoma.Previously researchers have reported significant loss of TM cells and obviously increased extracellular matrix in TM tissues of patients with primary open-angle glaucoma,compared with normal controls.So,we suggest that if the molecular mechanisms of glaucoma pathogenesis,such as TM cells proliferation, apoptosis and secretion,could be clearly elucidated,glaucoma could be effectively treated.Nuclear factor-κB(NF-κB) regulates the transcription of a wide array of gene products that are involved in many pathological and physiological pathways,such as cell differentiation,proliferation and apoptosis,and immune/inflammatory responses. Activation of NF-κB may be a pivotal event in proinflammatory signal transduction.But, its excessive activation would lead to many diseases with severe chronic/acute inflammatory responses.Inflammatory responses commonly occur during the glaucoma onset,development and treatment,especially in that caused by trauma and uveitis.High levels of inflammatory mediators and cytokines,produced in inflammatory responses in anterior chamber,would inevitably initiate the activation of NF-κB,which also could be induced by stress reactions.It have been found that the expression of NF-κB of TM cells in patients with primary glaucoma is significantly higher than that in normal controls. Unfortunately,the effects of NF-κB activation on glaucoma still remain unclear.NF-κB,critical to cell survival,could mediate anti-apoptosis and cell proliferation by regulating the expressions of relative genes.Additionally,it also participates in controlling MMP gene activity.In clinic,chronic administration of glucocorticoid could result in secondary glaucoma.Because glucocorticoid belongs to one of antagonists of NF-κB,we suggest that glucocorticoid abuse could heavily inhibit the activity of NF-κB and consequently induce TM cells apoptosis.However,no experimental evidences show that the cascade processes described above are responsible for the occurrence of glucocorticoidinduced glaucoma.In our research,to evaluate the effects of NF-κB on TM cells’ proliferation and apoptosis,and the transcription and expression of MMP and TIGR genes,we transfected RelA/P65 subunit gene into cultured TM cells and used oligodeoxynucleotides(ODNs), homologous to the sequence of cis-elements of NF-κB,to inhibit the activation of NF-κB in TM cells,according to the decoy strategy.At the same time,we tried to explain NF-κB role in TM cells dysfunction,and to evaluate the effect of different NF-κB activity on TM cells and its relationship with glaucoma occurrence.Materials and Methods:1.Primary culture and identification of TM cells:TM cells were cultured using tissue-explant technique with eye tissues of donors in corneal grafting.SP immunocytochemical method was used to identify fibronectin(FN),laminin(LN), CollagenⅣ,TIGRand FactorⅧ-related antigens.2.Cell grouping:The cells were divided into five groups,i.e.,normal control group (C),control plasmid group(P65-C),pRSV-RelA/P65 plasmid group(P65) and Random-decoy-ODNs control group(ODN-C) and NF-κB decoy-ODNs group(ODN)3.Transfection of P65 and NF-κB decoy-ODNs:After pRSV-RelA/P65 plasmid and NF-κB decoy-ODNs were transfected with liposome,the transfection efficiency was estimated by fluorescence microscope and Western Blot analysis.4.Detection of TNF-αlevel in pRSV-RelA/P65 plasmid transfected TM cells:At 24 hours after transfection,TNF-αlevel was measured by ELISA in the culture supematant of P65-transfected TM cells.5.Effect of NF-κB on TM cells’ proliferation and apoptosis:MTT assay and ~3H-TdR incorporation were applied respectively for the detection of TM cells’ proliferation,while Tunel method for their apoptosis. 6.Effect of NF-κB on the expressions of MMPs and their tissue inhinitors(TIMPs) in TM cells:RT-PCR was adopted to evaluate the expressions of mRNAs of MMPs(MMP-1, -2 and-3) and TIMPs(TIMP-1,-2 and-3).7.Effect of NF-κB on TIGR gene expression and regulation:The levels mRNA and protein expression of TIGR were detected respectively by RT-PCR and Western Blot.Results:1.We successfully cultured TM cells in DMEM.medium.Immunochemical analysis revealed that these cells were positive for LN,FN,CollagenⅣand TIGR gene expression, and negative for FactorⅧ-related antigens.2.At 24 hours after P65 gene transfection,the expression of P65 was dramatically leveled up,meanwhile,TNF-αlevel was also significantly increased in the culture supernatant of TM cells.3.Both relatively high expression of P65 and NF-κB decoy-ODNs could inhibit proliferation and DNA synthesis of TM cells,and consequently induce cell apoptosis.4.High P65 expression also had an evident expression-promoting effect on the mRNAs of MMP-1 and-3,while,NF-κB decoy-ODNs had an inhibitory one.At the same time,Both of them had no obvious effect on the expressions of MMP-2 and TIMP-1 and-2.5.The gene and protein expression of TIGR could be downregulated by High P65 expression,and upregulated by NF-κB decoy-ODNs.Conclusions:1.P65 obviously promotes TNF-αsecretion in TM cells,which indicates that excessive NF-κB activation may lead to the high-level inflammatory responses.2.Upregulated NF-κB expression would inhibit the proliferation and DNA synthesis of TM cells cultured in vitro and promote their apoptosis,which suggests that exuberant inflammatory responses in anterior chamber have a negative effect on TM cells survival and excessive activation of NF-κB may is one of the pathways involved in secondary glaucoma.NF-κB decoy-ODNs also suppress TM cells proliferation and DNA synthesis,and induce apoptosis,which suggests that NF-κB could maintain normal TM cell numbers and functions and chronic inhibition of NF-κB may cause glucocorticoid-induced glaucoma.NF-κB activation/inhibition could upregulate/downregulate the expressions of MMP-1 and-3 in TM cells,which indicates that NF-κB plays a critical role in ECM maintenance and chronic administration of NF-κB inhibitor may result in the decrease of secretion of MMP-1 and-3,and ECM accumulation in TM cells.NF-κB activation/inhibition also could cause changes of TIGR gene expression,which suggests that NF-κB plays a key role in regulating the expression of glaucoma-related TIGR gene and the inhibition of NF-κB function could lead to the increase of expression of TIGR protein in TM of glucocorticoid-induced glaucoma.更多还原