TPCK对大鼠晶状体混浊的影响及机制的研究
摘要
白内障是目前世界上影响人类健康的常见病，也位列我国第一位致盲的疾病，其发病机制尚不十分清楚。白内障的发生与多种因素有关，是多种因素综合作用的结果。比如晶状体蛋白的基因突变、晶状体遭受氧化损伤、辐射损伤、老化变性、葡萄糖过度刺激、半乳糖等代谢障碍、脂质过氧化损伤等均可对晶状体的正常功能造成不同程度的损伤，结果导致晶状体发生混浊，影响晶状体对光的透射和折射，从而造成视物不清。
氧化应激是由活性氧（ROS）诱导的一种与老化密切相关的损伤类型，与老年性白内障的形成有关，被认为是白内障的始发因素。晶状体产生的主要活性氧分子包括超氧负离子、羟自由基和H2O2；内源性产生的活性氧的结构包括NADPH氧化酶，线粒体和过氧化物酶体，两种主要产生内源性活性氧的途径是紫外线和离子辐射。晶状体中通过这些途径产生的ROS分子可以经由抗氧化剂和氧化防御系统中和，因此晶状体中具备这两大修复系统来重塑损伤分子或者减轻损伤程度。硫醇转移酶（thioltransferase, TTase）、硫氧还蛋白（thioredoxin, Trx）和硫氧还蛋白还原酶（thioredoxin reductase, TR）和谷胱甘肽还原酶（glutathione reductase, GR）系统被认为是哺乳动物组织中强有力的蛋白修复系统。近年的研究显示TTase/GR和Trx/TR均存在于晶状体中，对保持晶状体的透明性有重要的作用。
晶体蛋白是哺乳动物晶状体细胞质中的主要结构蛋白，根据其在电场中的迁移能力主要分为α、β、γ三大类。目前对α晶体蛋白的功能研究比较深入，它的分子伴侣作用已得到世界公认，γ晶体蛋白主要承担维持晶状体正常形态的作用，然而对β晶体蛋白的研究还较少，以往认为其与γ晶体蛋白一样作为晶状体的结构蛋白存在，近年来β晶体蛋白成为研究热点，我们前期研究表明了它对维持晶状体的透明性和屈光度有一定的作用。晶体蛋白中均含有一定数量的半胱氨酸（Cys, αB除外），尤其是β、γ类含量较高，Cys含一游离的巯基（-SH），对于氧化极为敏感；巯基经氧化作用形成二硫键混合物，是晶体蛋白去折叠、聚集增强的主要动因。由此产生一对矛盾：氧化应激会使巯基氧化，而为了保持晶状体的透光性，晶体蛋白必须维持足够量的还原状态的巯基（-SH）。
晶体蛋白的翻译后修饰（post-translational modifiction, PTM）是白内障发病的主要诱因。晶状体是一种结构极为特殊的器官：透明、缺乏血供，晶体蛋白缺乏转化更新、形成后即伴随终生。使晶体蛋白遭受PTM的可能性远较其它类蛋白大。PTM对晶体蛋白的主要影响是：极易引发晶体蛋白去折叠、改变蛋白原有构象；二硫键形成、晶体蛋白之间非特异交联，蛋白水溶性下降；聚集增强形成高分子量物质（High molecular weight, HMW），当HMW聚集达到1×107Da以上时，即可发生光散射，晶体失去透明性、发生白内障。
那么如何保持晶体蛋白中-SH未被氧化交联为二硫键，进而阻断后续蛋白分子交联、聚集、沉淀，对维持晶体透光性及防治老年性白内障起着关键的作用
本课题组前期的研究成果表明了二硫苏糖醇（dithiothreitol, DTT，分子式为C4H10O2S2）具有良好的抗晶状体蛋白变性的作用，DTT是一类化学性质的还原剂，它可以解开蛋白质分子内部及蛋白质分子间的二硫键，作为巯基的保护剂，能够抑制晶状体蛋白的热变性及非特异性聚集沉淀。课题组前期研究显示出在用自行配制的DTT滴眼液第8天后能使硒性白内障小鼠模型晶状体混浊的程度减轻。而且我们研究也显示出DTT对紫外线性白内障大鼠模型具有良好的抑制作用。DTT不仅可以防止蛋白质分子间形成二硫键，并且可以和蛋白质竞争性结合-SH，因此在预防和治疗方面都发挥了一定的作用。前期实验也初步验证了异丙醇对晶体蛋白巯基的烷基化作用，通过封闭部分巯基，抑制了二硫化合物的形成。
能够对蛋白质的巯基进行烷基化的试剂有很多，我们筛选出了一些化合物，例如叔丁醇(Tert-butyl alcohol, TAB)、2-SH-3-丁醇、N-对甲苯磺基-L苯乙胺酰氯甲基酮、甲苯磺胺氯甲基酮等等，其中叔丁醇对硒性白内障的治疗作用也得到了我们初步的证实。N-对甲苯磺基-L-苯乙胺酰氯甲基酮（N-tosyl-L-phenylalanyl-chloromethylketone, TPCK），研究已经证实能够抑制细胞的凋亡以及抗炎等作用对脑缺血有一定的治疗效果并初步应用于临床。对甲苯磺酰基（Tosyl, Ts）是一个由对甲苯磺酸衍生出的取代基，它是一个亲电子基，常用作醇羟基的保护基；而甲基酮作为烷化剂可以封闭晶体蛋白暴露出的巯基，抑制蛋白之间的聚合，因此可以阻止和延缓晶状体的混浊程度。
基于前期实验结果，我们首先将离体培养晶状体并模拟氧化应激的环境，加入TPCK干预后，观察TPCK对晶状体混浊程度的抑制作用，检测其中酶含量的变化，观察细胞凋亡的情况，研究TPCK抗氧化损伤的效果，研究其机制。
此外，泛素-蛋白酶体通路（ubiquitin-proteasome pathway, UPP）是Hershko等发现的一个高效率蛋白质降解系统，目前国内外对于UPP的研究主要集中在肿瘤的发病机制以及治疗学方面，是蛋白质的选择性降解中一项非常重要的机制；研究发现该通路介导的细胞蛋白降解是一个非常复杂而缜密的调控过程，是细胞调控的重要机制，通过降解细胞各通路的抑制因子和（或）激活因子发挥着上调或下调作用。最新有实验研究出晶状体上皮细胞中也含有UPP系统的所有组成部分，大约有40～50%受损的晶体蛋白可以通过UPP进行降解。UPP系统是晶状体中主要的蛋白质质量控制体系，它可以通过选择性地降解受损的晶状体蛋白，还能发挥有条件地降解与细胞内多种生物过程相关的调控蛋白的作用，参与晶状体的发育与分化。近年来，UPP作为细胞内蛋白质非溶解体性降解的主要途径已受到人们的日益重视，但UPP在白内障的研究目前仍处于起步阶段。
本课题第三部分内容研究UPP对晶状体上皮细胞内受损蛋白质水平的影响，探讨其是否能够清除受损变性的晶体蛋白。我们拟通过原代培养的晶状体上皮细胞，对UPP活性和白内障形成及受损蛋白之间的关系进行初步评估，以期得到抗白内障的作用机理。总之本课题为白内障药物防治的临床前期研究提供可靠的理论实验依据，而且这将对于其它年龄相关性眼部疾病的防治也有重要的参考价值。
第一部分TPCK对晶状体氧化应激下混浊程度和热稳定性的影响
目的：探讨TPCK在培养的大鼠离体晶状体中抵抗氧化应激的能力及对晶状体蛋白热稳定性的影响。方法：取完整晶状体用H2O2诱导其氧化损伤，分别在不同的时间点观察完整晶状体在氧化损伤组和用TPCK干预组的混浊程度。检测不同组的晶状体蛋白的热稳定性的变化；结果：未加入TPCK仅用H2O2诱导氧化损伤的晶体混浊程度高于TPCK用药组；晶状体蛋白吸光度值在用TPCK药物组低于氧化损伤组且上升速度缓慢。结论：TPCK能够在一定程度上减轻晶状体氧化应激状态下混浊程度的发生并稳定晶状体蛋白。
第二部分TPCK对晶状体氧化应激中抗氧化酶和凋亡的影响
目的：探讨H2O2诱导晶状体氧化损伤用TPCK干预后对抗氧化酶含量的影响及TPCK对细胞凋亡的作用。方法：同第一部分采用晶状体离体培养的方法加入H2O2引起氧化损伤，另加入TPCK干预，对照组晶状体仅用TC-199培养剂。在不同时间点分别取晶状体测定TTase、Trx的变化并观察TUNEL凋亡的情况；另原代培养晶状体上皮细胞（Lens epithelial cells, LES），紫外线诱导凋亡后观察TPCK的影响。结果：氧化应激后第3hr两组的TTase、Trx都有显著的提高，第6hr氧化损伤的晶状体中这两种酶的含量减少，TPCK组的TTase、Trx含量低于正常对照组但明显高于氧化损伤组，差别具有统计学意义；TUNEL凋亡提示，在培养3hr的晶状体上皮细胞中细胞凋亡在H2O2损伤组最多，TPCK组较少；细胞培养晶状体上皮细胞流式凋亡检测显示TPCK组细胞胞亡数少于氧化应激组。结论：TPCK可减少大鼠晶状体的氧化损伤时的程度。
第三部分UPP对晶状体上皮细胞内受损蛋白质水平的影响
目的：探讨过度表达UPP的限速成分Ubc4对晶状体上皮细胞内受损蛋白质水平的影响。方法：采用原代培养的方法体外培养晶状体上皮细胞，经UPP通路的上调因子miRNA Ubc4转染到传代的晶状体上皮细胞中，检测转染后的细胞对抗环境应激能力的影响。结果：转染Ubc4上调UPP的活性后的晶状体上皮细胞中受损的晶状体蛋白αA1-162（是αA晶状体蛋白的降解片段，容易聚集沉淀；αA晶状体蛋白是一种公认的保护性晶状体蛋白，该蛋白受损遭到降解后其保护作用降低）的含量明显少于未转染细胞中的水平。结论：转染siRNA Ubc4后的晶状体上皮细胞中的αA降解片段（αA1-162）含量减少，有助于防止受损蛋白质的聚集沉淀，进而在晶状体受到氧化应激的损伤时混浊变性具有一定的保护作用。
Cataract is still a main disease of growing blind in our country. And it influencedseriously the health of human all of the world at present which pathogenesis is not yet clear.Cataract is a multi-cause disease such as gene mutation, oxidative stress, radiation, age andso on. That reasons lead to lens opacity and further affect the lens transmission andrefraction of light that manifested in the symptoms is blurred vision.
Oxidative stress induced by reactive oxygen species (ROS) has long been implicatedin senile cataract formation. ROS molecules are generated in the lens include superoxideanion, hydroxyl radical, and H2O2. Endogenous systems that can generate ROS in the lensinclude NADPH oxidase, mitochondria, and peroxisomes. Two main sources that produceexogenous ROS are UV light and ionic radiation. ROS molecules produced through theseprocesses in the lens are neutralized by antioxidants and oxidation defense systems. Hence,the lens is also equipped with enzyme systems that can repair the damaged molecules1ordegrade the severely damaged ones. Thioredoxin (Trx)/thioredoxin reductase (TR) andthioltransferase (TTase)/glutathione reductase (GR) systems have been identified aspowerful protein repair systems in mammalian tissues. Recent work has shown that bothTTase/GR and Trx/TR systems are present in the lens and play a critical role inmaintaining the lens in a reduced state.
Lens proteins (crystallins) is the major structural protein components of the vertebrateeye lens, which are all built in the same manner and contains α-, β-, γ-crystallins accordingto migration in the electricfield. α-crystallins are mainly recognized to function aschaperones in the lens, and γ-crystallins basically maintain the lens chape as a structuralprotein, but little is known about β-crystallins. The regular structure and water-solubleingredient point to an important role for lens high refractive index and transparence thatmakes β-crystallins become a central issue in recent years. There are cysteine in crystallinswhich are extremely sensitive to oxidation due to the thiol. Adjacent thiol sufferingoxidation can form disulfide mixture that is the main reason of crystallins unfolding,aggregation. So lens could maintain transparent relying sufficient amount thiol.
Post-translational modification of crystallins is the chief incentives. CrystallinsPTM is more than other protein because its special struture that transparency, lack of bloodsupply, difficult to update. Crystallins water-soluble decline, aggregation enhanced result in high molecular weight which measure up to1×107Da could come cataract accompaniedby light scattering and opacity.
Therefor how to keep crystallins free-SH not be cross-linked to disulfide bonds play akey role in maintenance lens transparent and prevention of senile cataract.
Preliminary research results in our laboratory show that anti-lenses degeneration ofDTT（C4H10O2S2）is well. It is a chemical reductant that can untie disulfide linkage withinthe protein molecular and intermolecular. It also can inhibit the thermal denaturation oflens protein and non-specificity aggregation as sulfhydryl protective agent. Studies havebeen proved the nucleus plaque of selenite induced rat nuclear cataract after using DTT issignificant less than control group. Our data shows that DTT has obvious inhibitory actionon ultraviolet cataract. DTT can ont only prevent the formation of disulfide bonds betweenprotein molecules but also combine competitively-SH with protein, that can play a role inthe prevention and treatment. Our experiments validated the alkylation of isopropanol oncrystallins thiol so that inhibit the disulfide.
Many reagent can take alkylation effect of protein thiol such as tert-butyl alcohol,2-SH-3-butanol and TPCK. Study have been shown that TPCK can inhibit cell apoptosisand anti-inflammtory therefor that have a certain therapeutic effect on cerebral ischemia.Tosyl is a substituent derived from p-toluenesulfonic acid which used as protection ofalcoholic hydroxyl. And methyl as an alkylating can be closed exposed crystallins thiol,inhibit aggregation between proteins, and so it can prevent or delay lens opacity.
Thence we cultured lens in vitro and sumulate the environment of oxidative stress.Use TPCK to observe the inhibition of lens opacity, detect enzyme changes and checkapoptosis in order to research effect of TPCK on lens oxidative damage, study itsmechanism.
Ubiquitin-proteasome pathway (UPP) is a efficient protein degradation systemdiscovered by Hershko et al. which is related with the pathogenesis and treatment of cancerthat turn into hot content for domestic and foreign academic. Recent study found that UPPis a very complex and careful regulation system of protein degradation which play up-ordown-regulated role by degradation activator or inhibitor factor. Lens epithelial cellscontain all parts of UPP and about40-50%damaged cytosolic protein degradated by UPP.UPP is one of the proteolytic system that selectively degrade modified or damaged proteins which is related to regulation of biological process inside the cells participating lensdifferentiation. Recent years UPP become research hotspot as a important non-lysosomalpathway of intracellular protein degradation but the study about relationship with cataractis still initial stage.
This work the last one parts will assess the relationship of cataract and damagedprotein and UPP by means of cultured primary LEC based on latest research results inorder to understand the mechanism of anti-cataract drug and offer clinical evidence. It alsoprovide important theoretical value for other age-related eye disease prevention.
Part I The effect of TPCK on lens turbidity and thermal stability under oxidativestress
Objective: To investigate the role of TPCK in resistance to oxidative stress andthermal stability in rat lens culture. Method: Intact lenses were cultured in TC-199andthen observe opacity between the oxidative damage group and TPCK group at differenttime points. Detect thermal stability changes of crystallins protein in different groups.Result: The lens in H2O2oxidative damage not acceded TPCK more turbid than the TPCKgroup and the lens homogenates absorbance value lower than TPCK group and thecurvature result is the same trend. Conclusion: TPCK can mitigate lens turbidity underoxidative stress to a certain extent and satbilize the lens protein.
Part II The influence of TPCK on lens antioxidant enzymes and apoptosis
Objective: To investigate effects of TPCK in antioxidant enzymes and apoptosis inoxidative damaged lens. Method: Cultured rat lens in vitro same as the first part toresearch TTase and Trx contents at different time points and observe apoptosis. Thenculture lens epithelial cells to detect apoptosis after ultraviolet induced. Result: Theactivity of TTase and Trx significant improved after3hours but all reduced rapidly after6hours. The two enzymes in TPCK group are more than the oxidative damaged group butless than the control group. TUNEL results show that lens epithelial cells number is morein the H2O2oxidative damage group than TPCK group and flow cytometry apoptosis provethat apoptosis cells is obviously lower in the TPCK groups. Conclusion: TPCK can reducethe degree of oxidative damage in rat lens.
Part III The function of UPP on damaged protein in lens epithelial cells
Objective: To investigate effects of overexpressed Ubc4for impaired lens protein inlens epithelial cells. Method: Using cultured rat lens epithelial cells same as the first partto evaluate the ability of cells against environmental oxidative stress after transfectedup-regulation factor siRNA Ubc4. Result: The damaged αA1-162crystallin (is a degradationfragment which is easily precipitated of αA crystallin which is a protective protein in lensthat may reduce the role after suffering damage) level significantly reduced aftertransfected siRNA Ubc4which can up-regulate UPP activity compared withnon-transfected cells. Conclusion: The content of αA crystallin degradation fragment(αA1-162crystallin) after transfected siRNA Ubc4to lens epithelial cells which can preventdamaged lens protein from aggregation and precipitation and then this will play aprotective effect in lens under oxidative damage.
氧化应激是由活性氧（ROS）诱导的一种与老化密切相关的损伤类型，与老年性白内障的形成有关，被认为是白内障的始发因素。晶状体产生的主要活性氧分子包括超氧负离子、羟自由基和H2O2；内源性产生的活性氧的结构包括NADPH氧化酶，线粒体和过氧化物酶体，两种主要产生内源性活性氧的途径是紫外线和离子辐射。晶状体中通过这些途径产生的ROS分子可以经由抗氧化剂和氧化防御系统中和，因此晶状体中具备这两大修复系统来重塑损伤分子或者减轻损伤程度。硫醇转移酶（thioltransferase, TTase）、硫氧还蛋白（thioredoxin, Trx）和硫氧还蛋白还原酶（thioredoxin reductase, TR）和谷胱甘肽还原酶（glutathione reductase, GR）系统被认为是哺乳动物组织中强有力的蛋白修复系统。近年的研究显示TTase/GR和Trx/TR均存在于晶状体中，对保持晶状体的透明性有重要的作用。
晶体蛋白是哺乳动物晶状体细胞质中的主要结构蛋白，根据其在电场中的迁移能力主要分为α、β、γ三大类。目前对α晶体蛋白的功能研究比较深入，它的分子伴侣作用已得到世界公认，γ晶体蛋白主要承担维持晶状体正常形态的作用，然而对β晶体蛋白的研究还较少，以往认为其与γ晶体蛋白一样作为晶状体的结构蛋白存在，近年来β晶体蛋白成为研究热点，我们前期研究表明了它对维持晶状体的透明性和屈光度有一定的作用。晶体蛋白中均含有一定数量的半胱氨酸（Cys, αB除外），尤其是β、γ类含量较高，Cys含一游离的巯基（-SH），对于氧化极为敏感；巯基经氧化作用形成二硫键混合物，是晶体蛋白去折叠、聚集增强的主要动因。由此产生一对矛盾：氧化应激会使巯基氧化，而为了保持晶状体的透光性，晶体蛋白必须维持足够量的还原状态的巯基（-SH）。
晶体蛋白的翻译后修饰（post-translational modifiction, PTM）是白内障发病的主要诱因。晶状体是一种结构极为特殊的器官：透明、缺乏血供，晶体蛋白缺乏转化更新、形成后即伴随终生。使晶体蛋白遭受PTM的可能性远较其它类蛋白大。PTM对晶体蛋白的主要影响是：极易引发晶体蛋白去折叠、改变蛋白原有构象；二硫键形成、晶体蛋白之间非特异交联，蛋白水溶性下降；聚集增强形成高分子量物质（High molecular weight, HMW），当HMW聚集达到1×107Da以上时，即可发生光散射，晶体失去透明性、发生白内障。
那么如何保持晶体蛋白中-SH未被氧化交联为二硫键，进而阻断后续蛋白分子交联、聚集、沉淀，对维持晶体透光性及防治老年性白内障起着关键的作用
本课题组前期的研究成果表明了二硫苏糖醇（dithiothreitol, DTT，分子式为C4H10O2S2）具有良好的抗晶状体蛋白变性的作用，DTT是一类化学性质的还原剂，它可以解开蛋白质分子内部及蛋白质分子间的二硫键，作为巯基的保护剂，能够抑制晶状体蛋白的热变性及非特异性聚集沉淀。课题组前期研究显示出在用自行配制的DTT滴眼液第8天后能使硒性白内障小鼠模型晶状体混浊的程度减轻。而且我们研究也显示出DTT对紫外线性白内障大鼠模型具有良好的抑制作用。DTT不仅可以防止蛋白质分子间形成二硫键，并且可以和蛋白质竞争性结合-SH，因此在预防和治疗方面都发挥了一定的作用。前期实验也初步验证了异丙醇对晶体蛋白巯基的烷基化作用，通过封闭部分巯基，抑制了二硫化合物的形成。
能够对蛋白质的巯基进行烷基化的试剂有很多，我们筛选出了一些化合物，例如叔丁醇(Tert-butyl alcohol, TAB)、2-SH-3-丁醇、N-对甲苯磺基-L苯乙胺酰氯甲基酮、甲苯磺胺氯甲基酮等等，其中叔丁醇对硒性白内障的治疗作用也得到了我们初步的证实。N-对甲苯磺基-L-苯乙胺酰氯甲基酮（N-tosyl-L-phenylalanyl-chloromethylketone, TPCK），研究已经证实能够抑制细胞的凋亡以及抗炎等作用对脑缺血有一定的治疗效果并初步应用于临床。对甲苯磺酰基（Tosyl, Ts）是一个由对甲苯磺酸衍生出的取代基，它是一个亲电子基，常用作醇羟基的保护基；而甲基酮作为烷化剂可以封闭晶体蛋白暴露出的巯基，抑制蛋白之间的聚合，因此可以阻止和延缓晶状体的混浊程度。
基于前期实验结果，我们首先将离体培养晶状体并模拟氧化应激的环境，加入TPCK干预后，观察TPCK对晶状体混浊程度的抑制作用，检测其中酶含量的变化，观察细胞凋亡的情况，研究TPCK抗氧化损伤的效果，研究其机制。
此外，泛素-蛋白酶体通路（ubiquitin-proteasome pathway, UPP）是Hershko等发现的一个高效率蛋白质降解系统，目前国内外对于UPP的研究主要集中在肿瘤的发病机制以及治疗学方面，是蛋白质的选择性降解中一项非常重要的机制；研究发现该通路介导的细胞蛋白降解是一个非常复杂而缜密的调控过程，是细胞调控的重要机制，通过降解细胞各通路的抑制因子和（或）激活因子发挥着上调或下调作用。最新有实验研究出晶状体上皮细胞中也含有UPP系统的所有组成部分，大约有40～50%受损的晶体蛋白可以通过UPP进行降解。UPP系统是晶状体中主要的蛋白质质量控制体系，它可以通过选择性地降解受损的晶状体蛋白，还能发挥有条件地降解与细胞内多种生物过程相关的调控蛋白的作用，参与晶状体的发育与分化。近年来，UPP作为细胞内蛋白质非溶解体性降解的主要途径已受到人们的日益重视，但UPP在白内障的研究目前仍处于起步阶段。
本课题第三部分内容研究UPP对晶状体上皮细胞内受损蛋白质水平的影响，探讨其是否能够清除受损变性的晶体蛋白。我们拟通过原代培养的晶状体上皮细胞，对UPP活性和白内障形成及受损蛋白之间的关系进行初步评估，以期得到抗白内障的作用机理。总之本课题为白内障药物防治的临床前期研究提供可靠的理论实验依据，而且这将对于其它年龄相关性眼部疾病的防治也有重要的参考价值。
第一部分TPCK对晶状体氧化应激下混浊程度和热稳定性的影响
目的：探讨TPCK在培养的大鼠离体晶状体中抵抗氧化应激的能力及对晶状体蛋白热稳定性的影响。方法：取完整晶状体用H2O2诱导其氧化损伤，分别在不同的时间点观察完整晶状体在氧化损伤组和用TPCK干预组的混浊程度。检测不同组的晶状体蛋白的热稳定性的变化；结果：未加入TPCK仅用H2O2诱导氧化损伤的晶体混浊程度高于TPCK用药组；晶状体蛋白吸光度值在用TPCK药物组低于氧化损伤组且上升速度缓慢。结论：TPCK能够在一定程度上减轻晶状体氧化应激状态下混浊程度的发生并稳定晶状体蛋白。
第二部分TPCK对晶状体氧化应激中抗氧化酶和凋亡的影响
目的：探讨H2O2诱导晶状体氧化损伤用TPCK干预后对抗氧化酶含量的影响及TPCK对细胞凋亡的作用。方法：同第一部分采用晶状体离体培养的方法加入H2O2引起氧化损伤，另加入TPCK干预，对照组晶状体仅用TC-199培养剂。在不同时间点分别取晶状体测定TTase、Trx的变化并观察TUNEL凋亡的情况；另原代培养晶状体上皮细胞（Lens epithelial cells, LES），紫外线诱导凋亡后观察TPCK的影响。结果：氧化应激后第3hr两组的TTase、Trx都有显著的提高，第6hr氧化损伤的晶状体中这两种酶的含量减少，TPCK组的TTase、Trx含量低于正常对照组但明显高于氧化损伤组，差别具有统计学意义；TUNEL凋亡提示，在培养3hr的晶状体上皮细胞中细胞凋亡在H2O2损伤组最多，TPCK组较少；细胞培养晶状体上皮细胞流式凋亡检测显示TPCK组细胞胞亡数少于氧化应激组。结论：TPCK可减少大鼠晶状体的氧化损伤时的程度。
第三部分UPP对晶状体上皮细胞内受损蛋白质水平的影响
目的：探讨过度表达UPP的限速成分Ubc4对晶状体上皮细胞内受损蛋白质水平的影响。方法：采用原代培养的方法体外培养晶状体上皮细胞，经UPP通路的上调因子miRNA Ubc4转染到传代的晶状体上皮细胞中，检测转染后的细胞对抗环境应激能力的影响。结果：转染Ubc4上调UPP的活性后的晶状体上皮细胞中受损的晶状体蛋白αA1-162（是αA晶状体蛋白的降解片段，容易聚集沉淀；αA晶状体蛋白是一种公认的保护性晶状体蛋白，该蛋白受损遭到降解后其保护作用降低）的含量明显少于未转染细胞中的水平。结论：转染siRNA Ubc4后的晶状体上皮细胞中的αA降解片段（αA1-162）含量减少，有助于防止受损蛋白质的聚集沉淀，进而在晶状体受到氧化应激的损伤时混浊变性具有一定的保护作用。
Cataract is still a main disease of growing blind in our country. And it influencedseriously the health of human all of the world at present which pathogenesis is not yet clear.Cataract is a multi-cause disease such as gene mutation, oxidative stress, radiation, age andso on. That reasons lead to lens opacity and further affect the lens transmission andrefraction of light that manifested in the symptoms is blurred vision.
Oxidative stress induced by reactive oxygen species (ROS) has long been implicatedin senile cataract formation. ROS molecules are generated in the lens include superoxideanion, hydroxyl radical, and H2O2. Endogenous systems that can generate ROS in the lensinclude NADPH oxidase, mitochondria, and peroxisomes. Two main sources that produceexogenous ROS are UV light and ionic radiation. ROS molecules produced through theseprocesses in the lens are neutralized by antioxidants and oxidation defense systems. Hence,the lens is also equipped with enzyme systems that can repair the damaged molecules1ordegrade the severely damaged ones. Thioredoxin (Trx)/thioredoxin reductase (TR) andthioltransferase (TTase)/glutathione reductase (GR) systems have been identified aspowerful protein repair systems in mammalian tissues. Recent work has shown that bothTTase/GR and Trx/TR systems are present in the lens and play a critical role inmaintaining the lens in a reduced state.
Lens proteins (crystallins) is the major structural protein components of the vertebrateeye lens, which are all built in the same manner and contains α-, β-, γ-crystallins accordingto migration in the electricfield. α-crystallins are mainly recognized to function aschaperones in the lens, and γ-crystallins basically maintain the lens chape as a structuralprotein, but little is known about β-crystallins. The regular structure and water-solubleingredient point to an important role for lens high refractive index and transparence thatmakes β-crystallins become a central issue in recent years. There are cysteine in crystallinswhich are extremely sensitive to oxidation due to the thiol. Adjacent thiol sufferingoxidation can form disulfide mixture that is the main reason of crystallins unfolding,aggregation. So lens could maintain transparent relying sufficient amount thiol.
Post-translational modification of crystallins is the chief incentives. CrystallinsPTM is more than other protein because its special struture that transparency, lack of bloodsupply, difficult to update. Crystallins water-soluble decline, aggregation enhanced result in high molecular weight which measure up to1×107Da could come cataract accompaniedby light scattering and opacity.
Therefor how to keep crystallins free-SH not be cross-linked to disulfide bonds play akey role in maintenance lens transparent and prevention of senile cataract.
Preliminary research results in our laboratory show that anti-lenses degeneration ofDTT（C4H10O2S2）is well. It is a chemical reductant that can untie disulfide linkage withinthe protein molecular and intermolecular. It also can inhibit the thermal denaturation oflens protein and non-specificity aggregation as sulfhydryl protective agent. Studies havebeen proved the nucleus plaque of selenite induced rat nuclear cataract after using DTT issignificant less than control group. Our data shows that DTT has obvious inhibitory actionon ultraviolet cataract. DTT can ont only prevent the formation of disulfide bonds betweenprotein molecules but also combine competitively-SH with protein, that can play a role inthe prevention and treatment. Our experiments validated the alkylation of isopropanol oncrystallins thiol so that inhibit the disulfide.
Many reagent can take alkylation effect of protein thiol such as tert-butyl alcohol,2-SH-3-butanol and TPCK. Study have been shown that TPCK can inhibit cell apoptosisand anti-inflammtory therefor that have a certain therapeutic effect on cerebral ischemia.Tosyl is a substituent derived from p-toluenesulfonic acid which used as protection ofalcoholic hydroxyl. And methyl as an alkylating can be closed exposed crystallins thiol,inhibit aggregation between proteins, and so it can prevent or delay lens opacity.
Thence we cultured lens in vitro and sumulate the environment of oxidative stress.Use TPCK to observe the inhibition of lens opacity, detect enzyme changes and checkapoptosis in order to research effect of TPCK on lens oxidative damage, study itsmechanism.
Ubiquitin-proteasome pathway (UPP) is a efficient protein degradation systemdiscovered by Hershko et al. which is related with the pathogenesis and treatment of cancerthat turn into hot content for domestic and foreign academic. Recent study found that UPPis a very complex and careful regulation system of protein degradation which play up-ordown-regulated role by degradation activator or inhibitor factor. Lens epithelial cellscontain all parts of UPP and about40-50%damaged cytosolic protein degradated by UPP.UPP is one of the proteolytic system that selectively degrade modified or damaged proteins which is related to regulation of biological process inside the cells participating lensdifferentiation. Recent years UPP become research hotspot as a important non-lysosomalpathway of intracellular protein degradation but the study about relationship with cataractis still initial stage.
This work the last one parts will assess the relationship of cataract and damagedprotein and UPP by means of cultured primary LEC based on latest research results inorder to understand the mechanism of anti-cataract drug and offer clinical evidence. It alsoprovide important theoretical value for other age-related eye disease prevention.
Part I The effect of TPCK on lens turbidity and thermal stability under oxidativestress
Objective: To investigate the role of TPCK in resistance to oxidative stress andthermal stability in rat lens culture. Method: Intact lenses were cultured in TC-199andthen observe opacity between the oxidative damage group and TPCK group at differenttime points. Detect thermal stability changes of crystallins protein in different groups.Result: The lens in H2O2oxidative damage not acceded TPCK more turbid than the TPCKgroup and the lens homogenates absorbance value lower than TPCK group and thecurvature result is the same trend. Conclusion: TPCK can mitigate lens turbidity underoxidative stress to a certain extent and satbilize the lens protein.
Part II The influence of TPCK on lens antioxidant enzymes and apoptosis
Objective: To investigate effects of TPCK in antioxidant enzymes and apoptosis inoxidative damaged lens. Method: Cultured rat lens in vitro same as the first part toresearch TTase and Trx contents at different time points and observe apoptosis. Thenculture lens epithelial cells to detect apoptosis after ultraviolet induced. Result: Theactivity of TTase and Trx significant improved after3hours but all reduced rapidly after6hours. The two enzymes in TPCK group are more than the oxidative damaged group butless than the control group. TUNEL results show that lens epithelial cells number is morein the H2O2oxidative damage group than TPCK group and flow cytometry apoptosis provethat apoptosis cells is obviously lower in the TPCK groups. Conclusion: TPCK can reducethe degree of oxidative damage in rat lens.
Part III The function of UPP on damaged protein in lens epithelial cells
Objective: To investigate effects of overexpressed Ubc4for impaired lens protein inlens epithelial cells. Method: Using cultured rat lens epithelial cells same as the first partto evaluate the ability of cells against environmental oxidative stress after transfectedup-regulation factor siRNA Ubc4. Result: The damaged αA1-162crystallin (is a degradationfragment which is easily precipitated of αA crystallin which is a protective protein in lensthat may reduce the role after suffering damage) level significantly reduced aftertransfected siRNA Ubc4which can up-regulate UPP activity compared withnon-transfected cells. Conclusion: The content of αA crystallin degradation fragment(αA1-162crystallin) after transfected siRNA Ubc4to lens epithelial cells which can preventdamaged lens protein from aggregation and precipitation and then this will play aprotective effect in lens under oxidative damage.
引文
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