甜菜碱高半胱氨酸甲基转移酶调控同型半胱氨酸水平抑制白内障形成的分子机制研究

Betaine-homocysteine methyl transferase (BHMT) catalyzes the synthesis of methionine from betaine and homocysteine (Hcy). Thus, BHMT is playing a key role in Hcy homeostasis. Resent studies indicate that elevated levels of Hcy are closely associated with cataract formation. In addition, BHMT was found highly expressed in monkey lens nucleus, i.e. reaching 10% of the total nuclear protein. These studies implicte the potnetial role of BHMT in maintaining lens transparency via regulating methionine and Hcy homeostasis. Our previous studies showed that the BHMT protein level was significantly decreased in the aged nuclear cataract group vs. young group. Though, the pathological mechanisms of reduced BHMT in cataractogenesis are still unclear. The purpose of the present study is first to investigate the morphological and biological alterations of lens epithelial cells challenged by Hcy in conditions of either overexpression or knocking down the BHMT, and second is to test in ex vivo cultured rat lenses. This including cell proliferation, apoptosis, endoplasmic reticulum (ER) stress markers, the reactive oxygen species (ROS) formation and antioxidative response gene expression. We will also analyze the changes in protein expression using isobaric tag for relative and quantitation (iTRAQ) approach to illucidate the mechanisms of BHMT anti-oxidative effect at cellular level. We believe this study will shade light for us to better understand the role BHMT in maintaining lens transparancy and will also be important to find therapeutic targe for cataract prevention.

甜菜碱高半胱氨酸甲基转移酶(BHMT)是蛋氨酸(Met)循环中重要的酶, 可催化同型半胱氨酸(Hcy)重新甲基化，从而有效降低Hcy的浓度。研究显示Hcy与年龄相关性白内障的发生关系密切。我们前期研究发现， BHMT含量在高龄白内障晶状体核的水平较年轻组明显下调，但 BHMT降低与白内障形成的关系尚不清楚。本研究将利用人晶状体上皮细胞系(HELCs)BHMT过表达和BHMT shRNA转染的细胞模型观察Hcy对HELCs的氧化损伤程度、检测活性氧（ROS）含量、抗氧化酶的活性以及蛋白质表达、内质网应激反应蛋白表达，利用iTRAQ技术分析Hcy对BHMT转染细胞下游蛋白质的改变，明确BHMT参与抗氧化损伤的作用环节和机制；通过离体晶状体孵育，观察BHMT对经Hcy处理的HELCs凋亡、抗氧化酶的活性和蛋白质表达的影响，为探讨BHMT在白内障发病中的作用机制奠定重要的理论和实验基础。

我们前期的研究表明：晶状体蛋白质质谱检测发现甜菜碱高半胱氨酸甲基转移酶(BHMT)与人白内障的形成有一定的关系。我们认为: 由于BHMT是Hcy重新甲基化生成Met的重要酶，因此，BHMT在白内障晶状体中的表达减少可能影响了晶状体中Hcy的含量而使晶状体中ROS水平升高，诱发氧化应激反应，并导致谷胱甘肽的减少而诱发白内障。本课题基于以上思路开展研究，通过研究初步阐明了BHMT在抑制Hcy诱导的白内障形成的机制，为白内障形成机制提供了新的依据和研究思路，并为进一步防治措施提供新途径。. 本次研究成功构建过表达BHMT的慢病毒载体,并有效感染人晶状体上皮细胞,为进一步研究高Hcy环境诱导白内障发生发展中BHMT的作用提供生物学基础。通过细胞学实验，证实了高表达甜菜碱高半胱氨酸甲基转移酶基因对高HCY诱导的人晶状体上皮细胞氧化损伤有保护作用，通过动物实验，观察到BHMT在体外可有效防护高Hcy环境下大鼠晶状体氧化损伤, 减轻内质网应激反应，清除ROS, 阻止细胞凋亡, 减轻晶状体混浊度，证明了其在防治与Hcy有关的疾病方面的积极的作用。并通过人体手术标本，利用蛋白质组学研究发现了不同眼轴人白内障标本中不同蛋白质的表达，进一步证实了不同眼轴晶状体结构蛋白的影响，为下一步研究奠定了基础。