目的：通过建立硫醇转移酶(TTase)基因敲除小鼠模型，观察其晶状体形态和生化方面随年龄的改变，探讨TTase在晶状体氧化还原系统中的重要作用及参与年龄相关性白内障形成的机制。

方法：建立TTase基因敲除小鼠模型并进行基因型鉴定。裂隙灯观察TTase基因敲除型和野生型小鼠白内障随年龄的发生情况。检测小鼠晶状体中谷胱甘肽(GSH)含量。Western blot检测晶状体中氧化产物蛋白质二硫化物(PSSG)的变化。免疫共沉淀法鉴定形成PSSG的蛋白质。观察纯化的重组人晶状体TTase(RHLT)对PSSG的脱硫醇作用。

结果：TTase基因敲除型小鼠和野生型小鼠白内障的发生均随年龄而增加，且主要表现为核性白内障。在TTase基因敲除型小鼠中，白内障最早从4月龄开始发生，9月龄时最为显著； 野生型小鼠白内障最早从9月龄开始发生，12月龄时最为显著。两种基因型小鼠晶状体中GSH含量均随年龄增加而下降，9月龄TTase基因敲除型小鼠晶状体中GSH下降更为显著，且PSSG的表达明显高于野生型，主要表现为高分子聚合物。免疫共沉淀反应证实形成PSSG的蛋白质包含肌动蛋白(actin)和三磷酸甘油醛脱氢酶(GAPDH)，这种积聚的PSSG可被GSH还原，且与纯化的RHLT反应更有效。

结论：TTase基因敲除可以加速小鼠年龄相关性白内障的发生，这与晶状体中PSSG的积聚相关，且形成的PSSG可被TTase脱硫醇，证实TTase在防止年龄相关性白内障的发生中发挥重要作用。

AIM: To examine the morphological and biochemical alterations in the eyes of Thioltransferase knockout(TTase KO)mouse model as a function of age, and to explore the important function in redox homeostasis in the lens and in the age-related cataractogenesis.

METHODS: TTase KO model was established in this laboratory. TTase KO and WT mice were examined and the lens opacity was classified by using a slit lamp. Each lens was homogenized in lysis buffer and processed for measurement of glutathione(GSH)level. Examination of Protein-GSH mixed disulfides(PSSG)formation in the lens by Western blot analysis. Immunoprecipitation was used to identify the proteins formed PSSG. Dethiolation of lens proteins was carried out using purified recombinant human lens TTase(RHLT).

RESULTS: The slit lamp examination showed an age-dependent nuclear cataract development in both eyes of the WT and TTase KO mice. The onset of cataract was 4mo in the KO mice and 9mo in the WT mice. The GSH loss showed in both groups during aging and was prominent in the TTase KO mice after 9mo old. PSSG in the lenses of both groups showed progressive elevation, whereas the lenses of the KO group had a higher level of PSSG after 9mo. These GSH-conjugated proteins were confirmed as actin and glyceraldehyes 3-phosphate dehydrogenase(GAPDH)by immunoprecipitation and they could be eliminated when the homogenates were treated with RHLT.

CONCLUSION: The results showed that deletion of TTase gene in the mouse could lead to an early age-dependent cataract formation and the PSSG formation in these lenses appeared to link directly to lens opacity. The PSSG could be dethiolated by TTase. This data strengthens that TTase plays an essential role in maintaining lens clarity.

国家自然科学基金青年项目(No.81300743)