【作者】 宁波；

【导师】 张忠志；

【作者基本信息】 中国医科大学 ， 眼科学， 2009， 博士

【摘要】 目的青光眼是继白内障之后的第二位致盲眼病,原发性开角型青光眼（primaryopen-angle glaucoma,POAG）是青光眼的主要和常见类型之一,POAG的发病机制至今尚未明确,倾向于认为遗传因素是主要病因之一。1993年Sheffield等在一个青少年发病的POAG家系成员的DNA全基因组搜索中,发现了小梁网糖皮质激素诱导反应蛋白基因突变的。1997年,Polansky等在研究激素性青光眼时,发现了一个随着使用糖皮质激素作用时间的延长而表达增多的基因,它最初被命名为糖皮质激素诱导的小梁网反应蛋白（trabecular meshwork inducible glucocorticoidresponse protein,TIGR）基因,1997年Stone等的研究最终确定了TIGR基因是原发性开角型青光眼的致病基因,从此,对青光眼致病基因的研究开展迅速、普遍。同年,TIGR基因被基因命名委员会（Genome Database Nomenclature Committee,GDNC）正式命名为Myocilin（MYOC）基因。最近的研究发现:当小梁网的myocilin蛋白量过多（分泌过剩或降解减少）,可能阻塞小梁网房水外流导致房水流出阻力增加;突变形式的myocilin蛋白无法被分泌和降解,大量异常的myocilin蛋白沉积在小梁网细胞内质网或细胞外基质（extracellular matrix,ECM）,通过细胞凋亡和/或ECM重排,影响到房水外流,使眼内压升高。MYOC/TIGR基因受糖皮质激素调控,在糖皮质激素的诱导下表达水平显著上升,导致myocilin蛋白聚集;另外,有文献报道,糖皮质激素体外作用于小梁网细胞可以造成内质网及ECM重排,细胞的粘附、吞噬能力下降及细胞周期改变,由此可以看出,激素处理的体外培养的小梁网细胞其产生的病理生理变化与文献报道的POAG的小梁网细胞的病理改变极为相似,因此,我们采用地塞米松处理体外培养的小梁网细胞,以便制作出近似POAG发病的病理生理改变,并运用siRNA干扰技术探讨MYOC基因在POAG发病过程中的作用。方法1、大鼠小梁网细胞的培养及鉴定:将体外培养的3代小梁网细胞用于细胞鉴定及实验。细胞免疫组织化学鉴定:3代细胞达到融合以后,进行抗兔的纤维粘连蛋白（fibronectin,FN）、层粘连蛋白（laminin,LN）和神经元特异性烯醇化酶（neuron-specific enolase,NSE）的免疫细胞化学染色鉴定。2、地塞米松培养大鼠小梁网细胞、培养后MYOC基因表达量的改变及细胞凋亡率检测:3代的大鼠小梁网细胞,用终浓度为10-7M地塞米松的15%FBS培养基培养7天,观察地塞米松处理后的细胞数目、形态、凋亡率的变化情况,并运用Real-timePCR方法检测MYOC基因表达量的改变。3、检测MYOC基因作用:（1）MYOC突变分析:运用RT-PCR方法扩增MYOC基因的cDNA,测序,并进行突变分析。（2）siRNA干扰技术:设立三个组别,分别为空白组,空质粒干扰组及地塞米松处理组,运用siRNA干扰各个实验组的大鼠小梁网细胞,观察siRNA干扰后地塞米松处理组与正常细胞组比较后的细胞数目、形态、凋亡率的变化情况。结果1、大鼠原代小梁网细胞倒置显微镜观察结果组织块接种后5～10天,开始有细胞从组织块边缘向外生长或者在组织块周围见数个游离的细胞群落。显微镜下细胞形态多样:梭形,椭圆形,不规则形等,细胞体积较大。细胞形态介于上皮细胞和成纤维细胞之间。原代细胞生长缓慢,一般需要10～15天才能达到融合,形成单层贴壁细胞,融合以后细胞形态逐渐接近。2、传代的大鼠小梁网细胞倒置显微镜及透射电镜结果（1）倒置显微镜观察:传代的大鼠小梁网细胞形态一致,多呈梭形,类似成纤维细胞形态,融合以后细胞密度较高,细胞体紧密相连,可以形成漩涡状匍匐生长的单层贴壁细胞,高倍下可以看到细胞胞体较大,胞质丰富,胞体多突起。传代细胞生长较快,多在5～7天便可以融合。（2）透射电镜观察:大鼠小梁网细胞胞体多突起,表面微绒毛多见。细胞核呈圆形、椭圆形,位于细胞中央,边缘常见凹陷,核仁明显,核膜清晰,核内染色质呈细颗粒状分散不均匀,密度较高。胞质内溶酶体、内质网及线粒体丰富,并且含有大量的微管及微丝,且多在细胞核附近。3、培养细胞的免疫细胞化学染色鉴定结果（1）纤维粘连蛋白染色:小梁网细胞染色阳性,细胞质呈棕黄色,颗粒状,有些彼此相连呈网状。（2）层粘连蛋白染色:小梁网细胞染色阳性,细胞质呈棕黄色,颗粒状,也可彼此连接成网状。（3）神经元特异性烯醇化酶染色:小梁网细胞染色阳性,细胞质呈棕黄色,颗粒状,也可彼此连接成网状。4、细胞生长曲线:细胞增殖速度并不是按直线增加,到7天增殖速度开始减慢,细胞数目变为平台期。5、10^-7M地塞米松培养7天后细胞倒置显微镜及透射电镜结果（1）倒置显微镜:与未干扰前正常细胞相比,细胞密度降低,形态变化不大,但细胞有收缩的趋势。（2）透视电镜:细胞出现凋亡改变,包括细胞核固缩,细胞核内细胞质边集,细胞质浓缩,细胞器肿胀,微管微丝溶解甚多,核周可以出现空泡区域等等。6、地塞米松培养小梁网细胞的MYOC基因测序结果RT-PCR扩增出1872bp的cDNA片段,包括全长功能区的1509bp碱基序列,测序后的序列与GenBank数据库对比分析符合率＞99%,没有发现突变位点。7、正常细胞组、地塞米松处理组、地塞米松培养细胞siRNA干扰组MYOC基因表达量分析real-time PCR结果:（1）MYOC基因的干扰率最高可达85.8%。（2）正常小梁网细胞MYOC基因相对含量为约为（1.87±0.09）×10^-2pg（Mean±Std）;地塞米松培养的小梁网细胞MYOC基因表达相对含量约为（28.93±1.20）×10^-2pg（Mean±Std）;地塞米松培养组是正常细胞细胞组含量的15.47±0.70倍（Mean±Std）;两组细胞MYOC基因表达量对比*p＜0.05,差异有统计学意义。（3）地塞米松培养后siRNA干扰组MYOC基因的相对表达量为（5.22±0.66）×10^-2pg（Mean±Std）;Dex培养细胞对照组表达量为（28.32±0.70）×10^-2pg（Mean±Std）;两组数据进行t检验,p＜0.05,说明两者差异有统计学意义。8、正常组,地塞米松培养组及地塞米松培养细胞siRNA干扰后细胞组凋亡率结果:正常3代小梁网细胞的凋亡率为4.50±0.41%（Mean±Std%）;Dex培养的小梁网细胞的凋亡率为29.35±0.47%（Mean±Std%）;干扰后的Dex培养组细胞凋亡率为25.51±1.02%（Mean±Std%）;两两进行t检验,p＜0.05,每两组之间的差异都具有统计学意义。结论1、大鼠小梁网细胞可以作为MYOC基因体外研究的手段2、地塞米松不能导致体外培养的小梁网细胞的MYOC基因产生突变的变化,因此,激素性青光眼发病的MYOC基因途径不是通过突变产生的3、MYOC基因在激素性青光眼及POAG发病过程中都是通过介导小梁网细胞的凋亡起作用的;另外,微管微丝的减少导致细胞骨架蛋白重排,小梁细胞的移行能力、吞噬能力降低,导致小梁网组织房水外流能力降低,使得眼压升高,从而引起青光眼性的视神经及视野改变。4、siRNA技术未来将有可能成为青光眼基因治疗的一种手段。更多还原

【Abstract】 ForewordGlaucoma is an ocular disease and the second leading worldwide cause of blindness,after cataract.Primary open-angle glaucoma（POAG） is one of the main and common types of glaucoma.Although the mechanism of POAG is unclear,it is currently tempting to argue that genetic factor is one of its main causes.In the whole field of glaucoma,there have been dozens of glaucoma-associated genes reported in succession,among which 15 chromosomal loci designated have been defined for POAG,but the candidate Disease-causing genes identified for POAG are still only MYOC / TIGR gene and OPNT gene.The role of Myocilin protein in POAG pathogenesis is still unclear.According to the present findings,we summarized its possible mechanism:The research on the expression of the normal and mutational MYOC / TIGR gene in ocular cells indicates that normal myocilin protein can be secreted,but almost any myocilin protein secretion expressed by various kinds of mutation form of MYOC/TIGR gene is not detected.The study that mutational myocilin protein deposited in endoplasmic reticulum is poisonous to the cells of human trabecular meshwork,shows that most mutations of MYOC / TIGR genes,such as Gly364Val,Gln368Stop,Lys423Glu etc.,cause the myocilin protein to stop secreting,and it have been determined that these mutational myocilin proteins accumulated in the endoplasmic reticulum lead to the weakening of cell proliferation and the subsequent dysfunction of trabecular meshwork cells until apoptosis,which is also considered as the cause of glaucoma.Because it is similar to the primary open-angle glaucoma in many aspects,such as pathophysiological process,the corticosteroid-induced glaucoma is likely to become the disease model for study of POAG.It has been established in the literature that there is similarity between the effects of dexamethasone on MYOC gene of trabecular meshwork cells in vitro and the ones in vivo.Therefore,we use dexamethasone to cultivate the trabecular meshwork cells of rat in vitro to simulate the pathogenesis process of POAG and determine the role ofMYOC gene in the pathogenesis process of POAGExperimental methods1.siRNA interference:According to the sequence of Myoc gene of rat（GenBank Accession Number:NM-030865）,select two loci to design siRNA,the sequence is shown in Table 1.2.After siRNA interference,the quantitative analysis of MYOC gene:We used 15%FBS medium containing 10^-7M dexamethasone（sigma） to cultivate the trabecular meshwork cells of the normal rat for 7 days,then carried out MYOC gene interference and tested the MYOC gene expression and the changes of the apoptosis rate of the trabecular meshwork cells.3.Data Analysis:All datas in experiments are treated by SPSS 11.5 Statistical Analysis Software.Experimental results1.The trabecular meshwork cells of rat cultivated by 15%FBS medium containing 10^-7M dexamethasone:After the trabecular meshwork cells of rat are cultured by 10^-7M dexamethasone medium for 7 days,the cell morphology is almost not changed,but the cell density is obviously lower,the change of apoptosis in the cell is seen,including karyopyknosis, Chromatin margination,cytoplasm condensation,organelle swelling,many microtubule-microfilaments are dissolved,perinuclear vacuolization region occur etc.2.siRNA interfered resulting picture:Using Real-time PCR method to do the relative quantitative analysis,and 18sRNA as a reference,the relative content ofMYOC gene of normal trabecular meshwork cells is about（1.87±0.09）×10^-2pg（Mean±Std）;the relative content of MYOC gene expression of trabecular meshwork cells cultivated in dexamethasone is about（28.93± 1.20）×10^-2pg（Mean±Std）;the content in the dexamethasone cultivating group is 15.47±0.70 times（Mean±Std ） of the one in the normal cell group;the comparison rate of MYOC gene expressive amount between the two groups of cells is p＜0.05,and the difference has the statistical significance.Using Real-time PCR method to do the relative quantitative analysisa,and 18sRNA as a reference,the interference rate available of Myoc-1242 and Myoc-1335 group are more than 80%,up to 85.8%,while all interference rates in other three groups are less than 75%,which can not reach the interference effect.Interference ratio =（mRNA amount in the comparison group-mRNA amount in the experimental group） / mRNA amount in the comparison group.The MYOC gene expression analysis results of trabecular meshwork cells cultivated in 10-7M dexamethasone after interference:the relative expression amount of MYOC gene cultivated in 10-7M dexamethasone in siRNA interference group is （5.22±0.66）×10-2pg（Mean±Std）;The expression amount in control group is（28.32±0.70）×10-2pg（Mean±Std）;The T test was done between the two groups of data, p＜0.05,and the difference has the statistical significance.3.The apoptosis test results of trabecular meshwork cells:The apoptosis rate of trabecular meshwork cell of normal 3rd generation of rat is 4.50±0.41%（Mean±Std%）;The apoptosis rate of trabecular meshwork cell of rat culivated in 10-7M dexamethasone is 29.35±0.47%（Mean±Std%）;The apoptosis rate of trabecular meshwork cell of rat cultured in dexamethasone after interference is 25.51±1.02%（Mean±Std%）;The T test was done between the two groups of data, p＜0.05,and the difference has the statistical significance.ConclusionMYOC gene is acting in the pathogeneses of steroid-induced glaucoma and POAG by mediating the apoptosis of trabecular meshwork cells;In addition,the reduction of microfilaments-microtubules leads to the rearrangement of cytoskeletal proteins and the weakened migrating ability of trabecular meshwork cells,which decreases the outflow ability of aqueous humor of trabecular meshwork,increases intraocular pressure,eventually causes the glaucomatous changes of the optic nerve and visual fields.更多还原