【作者】 谭健；

【导师】 姚克；

【作者基本信息】 浙江大学 ， 眼科学， 2007， 博士

【摘要】 白内障是世界范围内的主要致盲原因，目前还没有药物或者营养性的治疗可以消除已经存在的白内障或者延缓其进展，通过手术摘除白内障是主要的治疗方法。但由于植入人工晶体后失去调节能力以及存在发生后发性白内障（after cataract，或称为后囊膜混浊，posterior capsular opacification，PCO）和眼内炎症反应的可能，等等问题的出现，都在一定程度上影响了治疗的效果。更需要指出的是，发生PCO和需要行二次激光手术的患者主要是年轻人，尤其是儿童，以及糖尿病性视网膜病变或者外伤性白内障的患者。因此，研究明确白内障以及后发性白内障的发病机理，从而寻求更为安全有效的非手术的防治方法是非常重要的。转化生长因子β（TGF-β）是晶状体在各种生理和病理状态下重要的调节因子，与囊膜下型白内障以及后发性白内障的形成有密切关系。研究发现，TGF-β能够干扰正常晶状体结构并诱导晶状体上皮细胞发生异常的生长和分化并发生凋亡，导致体外培养的大鼠晶状体发生前囊膜下浑浊；而且，对人晶状体组织分析的大量数据也证明了TGF-β的这种作用。另外，TGF-β能够诱导后发性白内障的形成也被众多的研究所证明。TGF-β能够诱导含有平滑肌肌动蛋白的间叶细胞形成，晶状体囊膜皱缩，以及细胞外基质（包括laminin，fibronectin和Ⅰ型胶原等）的沉积。近期的研究还证明了在白内障术后，不仅TGF-β及其受体在晶状体囊袋内有表达，而且很多由TGF-β调节的蛋白，包括连接组织生长因子（connective tissue growth factor，CTGF）等均有明显表达。转化生长因子β（Transforming Growth Factorβ，TGF-β）在人体内发现的有3种同亚基二聚体，即TGF-β₁、TGF-β₂和TGF-β₃，以及一种异亚基二聚体TGF-β₁₂。其中，TGF-β₂在眼内前房及玻璃体内的含量及活性明显高于其他亚型，是诸多参与调节细胞生理和病理过程的细胞因子中最重要的。TGF-β₂是位于1q41染色体由特异基因编码的约25 kDa的多肽。该二聚体多肽广泛分布于人体内，并由多种不同细胞所合成。眼部的泪液、玻璃体和前房内都检测到它的存在。TGF-β₂在组织修复和产生细胞外基质中发挥重要作用。它能够抑制细胞增殖并发挥各种免疫抑制作用。在前房内，TGF-β₂通过改变抗原呈递细胞的活性，抑制T细胞增殖、IFN-γ的产生和巨噬细胞的免疫活性来发挥着维持免疫抑制状态的作用。TGF-β₂由角膜内皮细胞、小梁网细胞和睫状体分泌，以无活性的前复合体（latent TGF-β₂ complex，L TGF-β₂）形式存在。该前体（200 kDa）与潜在相关肽（latency-associated peptide，LAP）结合在一起与潜在TGF-β结合蛋白（TGF-βbinding protein，LTBP）相连接。只有当LAP和LTBP通过蛋白裂解后，L-TGF-β₂才能同其受体结合，其具体机制尚未完全阐明。其中一种活化的机制是L-TGF-β₂由细胞外基质蛋白酶释放后，通过thrombospondin-1或者整合素家族定位于细胞表面并活化。活化后的TGF-β₂通过结合其细胞膜内面的受体发挥生物学效应。各种眼球疾病均能够引起房水中TGF-β₂水平发生不同程度的改变，TGF-β₂的活化比例约占11％—61％。在白内障患者中，Tripathy et al.的研究发现，活化的TGF-β₂水平可以达到200±240 pg／ml（20-830 pg／ml）；Jampel et al.发现活化TGF-β₂占总TGF-β₂的61％。前期的研究都说明，TGF-β₂在诱导前囊膜下白内障形成和促进后发性白内障形成过程中发挥着重要作用。随着TGF-β₂对晶状体作用的研究不断深入，TGF-β₂发挥作用的机理受到广泛的重视和研究，本课题主要从研究TGF-β诱导晶状体上皮细胞凋亡的机理入手，发现和分析了活性氧在该过程中发挥的重要调控作用；在此研究的同时，我们发现TGF-β₂处理后，仍有大部分晶状体上皮细胞没有发生凋亡，表现出对TGF-β₂处理的耐受并发生明显的侵袭，我们对这一现象进行了分析后发现，整合素β₁及其介导的蛋白激酶通路在该过程中作用明显。在总结了以往关于TGF-β与白内障相关性研究的基础上，本研究就TGF-β₂对晶状体在各种生理病理状态下的影响进行了分析，对于全面阐明该细胞因子对晶状体的作用和作用机理做出了积极的工作。第一部分转化生长因子β₂诱导的人晶状体上皮细胞凋亡——活性氧的调节作用【研究目的】研究发现，转化生长因子β₂（transforming growth factorβ₂，TGF-β₂）能够抑制人晶状体上皮细胞（human lens epithelial cells，HLECs）的增殖并诱导细胞发生凋亡，该过程与晶状体前囊膜下浑浊密切相关，但是这两种过程中的信号传导机制仍不明了。活性氧（reactive oxygen species，ROS）作为细胞内重要的信号传导分子参与了众多的细胞损伤过程，因此，我们的本部分的研究目的是研究ROS在TGF-β₂对HLECs的损伤过程中的作用，进而明确TGF-β₂对HLECs的损伤机制。【研究方法】体外培养的人晶状体上皮细胞株HLE B-3，用不同浓度的TGF-β₂处理后，分别采用MTT和TUNEL法分析TGF-β₂对细胞增殖和凋亡的影响。应用荧光染料DCFH-DA，测定细胞内活性氧的含量改变；并且，我们测定了细胞还原型谷胱甘肽（GSH）的含量以及采用实时荧光定量RT-PCR法测定了c-fos基因的mRNA含量的变化。最后，我们通过应用不同的活性氧清除剂，抑制细胞内活性氧的升高，测定对TGF-β₂诱导细胞增殖和凋亡两个过程的影响。【研究结果】TGF-β₂是晶状体上皮细胞重要的生长调节因子，并且可以引起细胞的凋亡。量效研究发现100 pg／ml TGF-β₂将引起细胞凋亡，而10 pg／mlTGF-β₂即可抑制细胞的增殖。在100 pg／ml TGF-β₂引起HLECs凋亡的早期发现细胞内ROS含量明显增高，并且降低了细胞内GSH含量，说明TGF-β₂可以引起对HLECs的氧化性损伤。我们同时在研究c-fos这一受氧化状态调节的重要基因的mRNA表达中发现，只有高浓度的TGF-β₂（100 pg／ml）可以引起该基因的表达增高，其表达的变化情况和细胞经过氧化氢处理后的情况相似。最后，应用活性氧清除剂可以明显降低甚至阻止细胞发生凋亡，但是不能改善细胞增殖受抑制的状态。【研究结论】TGF-β₂能够明显抑制HLECs的增殖；并在达到一定浓度后诱导HLECs发生凋亡。ROS参与了TGF-β₂诱导凋亡的过程。第二部分转化生长因子β₂诱导的人晶状体上皮细胞移行和粘附——整合素β₁及其相关的蛋白激酶通路的作用【研究目的】在第一部分内容的基础上我们发现，TGF-β₂处理后，仍有大部分晶状体上皮细胞没有发生凋亡，表现出对TGF-β₂处理的耐受并出现出一定的侵袭现象。结合目前的其他研究的结果，白内障囊外摘除术后残留的晶状体上皮细胞是形成后发性白内障的重要原因，TGF-β₂也是参与该过程并起重要调节作用的细胞因子之一。因此，本部分的研究在第一部分的基础上，进一步发现了TGF-β₂对HELCs粘附和移行能力的影响，重点研究整合素β₁，这一在调节细胞粘附和移行方面重要的细胞表面分子的作用，以及整合素相关的粘着斑激酶（focal adhesion kinase，FAK）的活化情况。进一步分析和阐明了TGF-β₂促进后发性白内障形成的机理。【研究方法】培养的人晶状体上皮细胞株HLE B-3，用100 pg／ml的TGF-β₂处理后，检测细胞粘附和移行的改变。通过激光共聚焦及流式细胞仪测定细胞内整合素β₁亚型的蛋白表达，应用实时荧光定量RT-PCR法检测整合素β₁的mRNA的表达。用Western-blot法分别测定细胞内总FAK及其磷酸化Tyr 576蛋白的表达情况。最后，使用抗整合素β₁的单克隆抗体阻断细胞内整合素β₁后，检测对细胞粘附和移行的影响。【研究结果】TGF-β₂可以引起HLECs的凋亡，但仍有＞70％的细胞存活下来。TGF-β₂的处理能够引起该部分细胞粘附和移行力随着时间的延长明显升高；同时，细胞内整合素β₁的表达明显升高（P＜0.05）；其相关的FAK的磷酸化水平明显升高（P＜0.05）。应用抗整合素β₁的单克隆抗体预处理细胞后，能够明显抑制TGF-β₂引起的细胞粘附和移行。【研究结论】TGF-β₂能够明显促进HLECs的粘附和移行；整合素β₁及其相关的FAK的磷酸化参与了该过程并发挥重要作用。转化生长因子β₂（TGF-β₂）是在眼内起主要作用的TGF-β亚型，它对晶状体上皮细胞在生理和病理情况下具有多种调节作用。本课题研究发现，TGF-β₂能够抑制体外培养的晶状体上皮细胞的增殖，表现出一定的时效和量效关系；更重要的是它能够诱导晶状体上皮细胞发生凋亡，证明了TGF-β₂与某些白内障的形成存在关系。活性氧作为细胞内重要的调节因素，参与TGF-β₂诱导的晶状体上皮细胞的凋亡。在TGF-β₂诱导细胞凋亡的同时，我们发现仍有大部分细胞保持存活。我们对其细胞膜表面粘附分子的检测发现，TGF-β₂促进了整合素β₁的表达以及其相关的粘着斑激酶的磷酸化，通过该途径，TGF-β₂明显增加了晶状体上皮细胞的粘附率和移行能力，该结果进一步发现了TGF-β₂促进后发性白内障形成的机理。更多还原

【Abstract】 Cataract is the leading cause of blindness worldwide. There are still no medicine or threptic methods which can cure or delay the progress of cataract.The treatment for cataract is routine, surgical removal of cataracts and implantation of replacement lenses so far. But there are still many questions after the operation, such as loss of accommodation; after cataract and the possibility of intraocular inflammation, which would degrade the therapeutic effect. It is important to point out that the case for further treatment are always young especial children, and those who have diabetic retinopathy or traumatic cataract. It is important to clarify the mechianism of cataract and after cataract for searching more safe and effective therapeutic methods.Transforming growth factor β （TGF-β） has recently been identified as a criticalregulator of many pathological growth conditions in the lens. For example, TGF-β has been shown to induce anterior sub-capsular cataract （ASC） in a rat lens culture model and analysis of human tissue has created a body of evidence that strongly implicate TGF-β in this process also. TGF-β is also now being examined as a causative factor in another growth condition of the lens. Posterior capsule opacification （PCO）, which arises from vigorous lens cell growth following cataract surgery. Recent work has provided useful evidence that not only TGF-β and its receptors are present in capsular bags following surgery, but also some proteins which are strongly regulated by TGF-β, including connective tissue growth factor （CTGF）.At least five isoforms of TGF-β have been reported with TGF β- 1, 2 and 3 being present in mammals. In the eye, altered intraocular fluid concentrations of TGF-β have been reported in the context of various ocular conditions and pathologies. The major isoform synthesised within the eye is TGF-β₂. In addition to TGF-β₂ two further isoforms of TGF-β have been identified, TGF-β₁ and TGF-β₃. Both isoforms play an only minor role in immunomodulation of the anterior ocular segment.Transforming growth factor-β₂ （TGF-β₂） is an approximately 25 kDa polypeptide encoded by a unique gene located on chromosome lq41. This dimeric peptide is ubiquitously distributed in human tissues and synthesized by many different human cells. It has been detected in tear fluid, in the vitreous, and in aqueous humor. TGF-β₂ is known to play an important role in wound healing and the production of the extracellular matrix. It inhibits cell proliferation and exerts various immunosuppressive effects. In the anterior chamber, TGF-β₂ is relevant for the maintenance of an immunosuppressive climate, as it alters the activities of antigen-presenting cells, and suppresses T-cell proliferation, IFN-γ production, and the inflammatory activity of macrophages. It is secreted as an inactive precursor （latent TGF-β₂ complex, LTGF-β₂） by cell types such as corneal endothelial cells, cells of the trabecular meshwork, and the ciliary body. This precursor （200 kDa） is complexed with latency-associated peptide （LAP） and bound to latent TGF-β binding protein （LTBP）. L-TGF-β₂ is not able to bindto its receptor until LAP and LTBP are removed extracellularly via proteolytic cleavage. The exact mechanisms by which latent TGF-β₂ is activated physiologically are not completely understood. One model of activation has been proposed in which latent TGF-β is released from the extracellular matrix by proteases, localized to cell surfaces, and activated for example by thrombospondin-1 or specific integrins. Following this activation, TGF-β₂ exerts its biological functions via binding to a membrane-bound heteromeric receptorDifferent levels of total TGF-β₂ have been found in human aqueous humor, depending on the ocular disorders concerned. The ratio of active to total TGF-β₂ in the aqueous humor has been reported to be between 11 to 61% Tripathy et al. found active TGF-β₂ levels in cataract patients of 200±240 pg/ml （range 20-830 pg/ml）, and Jampel et al. found a ratio of active-to-total TGF-β₂ of 61% for cataract patients.Previous work have revealed that TGF-β play an important role in ASC and PCO. The mechanisms about the effect of TGF-β on lens are studied extensively. In this study, we started with the investigation of aoptosis in human lens epithelail cells induced by TGF-β₂. We found that reactive oxygen species （ROS） mediated this process and a majority of cells kept an vigorous acvitity to invasiveness. A further research found that integrin β₁ took part in this process. This study performed a deeply investigation about the effect of TGF-β on human lens epithelail cells from two aspects. All these work will benefit for reveal the mechanism about the effect of TGF-β on lens in and physiological and pathological conditions.Reactive oxygen species （ROS） mediate the apoptosis induced by transforming growth factor beta 2 in human lens epithelial cells[Objective]To investigate the possible role of reactive oxygen species （ROS） in the apoptotic process induced by transforming growth factor beta 2 （TGF-β₂） in human lens epithelial cells （HLECs）.[Methods]HLE B-3 cells were treated with different concentrations of TGF-β₂. MTT assay and TUNEL assay were used to analyze cell proliferation and apoptosis, respectively. We used DCFH-DA, an oxidation-sensitive fluorescent probe to examine the generation of ROS in HLECs that were treated with TGF-β₂. Furthermore, we investigated cells for glutathione and c-fos mRNA intracellular levels.[Results]Transforming growth factor-β₂, a growth regulator of HLECs in culture, also regulates the death of these cells. Dose-response analysis showed that the TGF-β₂ concentration needed to induce HLEC death （100 pg/ml） was 10 times that needed to inhibit growth in these cells （10 pg/ml）. TGF-β₂-induced apoptosis in HLECs was preceded by an induction of ROS and a decrease in glutathione in the intracellular content, indicating that this factor induces oxidative stress in HLECs. Studies performed to analyze the levels of c-fos mRNA, a gene whose expression is modulated by the redox state, demonstrated that only high, apoptotic concentrations of TGF-β₂ （100 pg/ml） produced an increase in the mRNA levels of this gene, the level of induction being similar to that found when cells were incubated in the presence of hydrogen peroxide. Finally, the cell death induced by TGF-β₂ in HLECs was partially blocked by radical scavengers, which decreased the percentage of apoptotic cells,whereas these agents did not modify the growth-inhibitory effect elicited by TGF-β₂ in these cells.[Conclusion]The results presented in this paper provide evidence for the involvement of an oxidative process in the apoptosis elicited by TGF-β₂ in HLECs.Part IIIntegrin β1 and Integrin-related signaling are necessary for transforming growth factor beta 2-promoted invasiveness inhuman lens epithelial cells[Objective]In this study, we investigate the possible implication of integrin and integrin-related signaling in TGF-β₂-promoted invasiveness in human lens epithelial cells.[Methods]A human lens epithelial cell line （HLE B-3） was treated with 100 pg/ml TGF-β₂ for 6, 12, 24h respectively. In vitro wound healing assay and cell adhesion assay were perfromed to detected the effect of TGF-β₂ on HELCs adhesion and migration. Integrin β₁ expression changes in HELCs during the treatment of TGF-β₂ were detected in protein level and mRNA level using confocal microscopy, flow cytometric analysisand real time quantitative reverse transcription polymerase chain reaction （RT-PCR） respectively. Focal adhesion kinase （FAK） activity was examined by FAK phosphorylation （Tyr 576） and total tyrosine phosphorylation during treatment with TGF-β₂ in HLEC. [Results]In this study, we found that TGF-β₂ significantly stimulated cell adhesion and migration in HLECs. By immunofluorescence staining and Western blotting, we observed that TGF-β₂ markedly enhanced the expression of integrin β₁ and the Tyr-Phosphorylation of focal adhesion kinase （FAK）. Real time quantitative RT-PCR also showed the mRNA level of integrin β₁ upregulated. Neutralizing anti-integrin β₁ monoclonal antibody inhibited TGF-β₂-promoted HLECs adhesion and migration significantly（P<0.05）.[ Conclusion ]TGF-β₂ promoted HLECs adhesion and migration in vitro. Integrin β₁ and integrin-related signaling are necessary for TGF-β₂-promoted adhesion and migration in human lens epithelial cells.SummaryThe multifunctional growth factor transforming growth factor β （TGF-β） is one of the most important ligands involved in modulation of cell behavior in ocular tissues. TGF-β, especially TGF-β₂, which is the predominant cytokine, involved in the regulation of cell behavior in lens in physiological or pathological processes of development or tissue repair, although various other growth factors are also involved. In this article, we found that TGF-β₂ induced supression of human lens eipthelail cells in time and concentration manner. Moreover, it induced cells underwent apoptosis which correlated with some types of cataract formation. Reactive oxygen species involved in and modulated this process.Furthermore, we found that a majority of cells survived. We found that TGF-β₂ stimulated integrin β₁ expression and the phosphorylation of focal adhesion kinase, which is the downstream of integrin signal. TGF-β₂ promoted human lens epitheial cells adhesion and migration through this signal. The results presented made a progress to clarify the mechinasm of TGF-β₂ induced posterior capsular opacification formation.更多还原