高糖对人晶状体上皮细胞硫醇转移酶表达的影响

Effects of high glucose on thioltransferase expression in human lens epithelial cells

目的观察高浓度葡萄糖对人晶状体上皮细胞硫醇转移酶(thioltransferase,TTase)表达的影响。方法将体外培养的人晶状体上皮细胞分别用葡萄糖浓度为5.5 mmol·L-1(正常对照组)、15.5 mmol·L-1、25.5 mmol·L-1及35.5 mmol·L-1(不同浓度高糖组),胎牛血清体积分数均为15%的DMEM培养液培养,同时设5.5 mmol·L-1葡萄糖+30.0 mmol·L-1甘露醇高渗对照组,24 h后Western-blotting检测TTase表达情况。选择35.5 mmol·L-1高糖浓度培养细胞,分别在0 h、2 h、8 h、16h、24 h时间点收集细胞,Western-blotting检测TTase表达,并测定TTase活性,细胞内总谷胱甘肽(T-GSH)、还原型谷胱甘肽(GSH)与氧化型谷胱甘肽(GSSG)含量,计算GSSG与T-GSH的比值。结果 TTase表达随葡萄糖浓度增加逐渐增强,在35.5mmol·L-1高糖组中表达最强,约为正常对照组的2.3倍(P<0.01),25.5 mmol·L-1高糖组约为正常对照组1.7倍(P<0.05),甘露醇高渗对照组TTase表达与正常对照组相比无明显变化(P>0.05)。在35.5 mmol·L-1高糖处理后2 h,TTase表达开始增高,8 h表达达到高峰,约为正常对照组的2.0倍(P<0.01),随后逐渐下降,16 h时约为正常对照组1.7倍(P<0.01);24 h降为1.5倍(P<0.05)。与TTase表达类似,TTase活性在35.5 mmol·L-1高糖处理后2 h开始升高,8 h达到高峰,约为正常对照组的1.8倍(P<0.01),随后逐渐下降。GSH在35.5 mmol·L-1高糖处理后16 h较对照组明显下降(P<0.05)。GSSG和GSSG/T-GSH比值在35.5 mmol·L-1高糖处理2 h后均显著升高,分别约为正常对照组的3.5倍(P<0.001)和3.3倍(P<0.001),并随时间推移不断上升。结论高糖可诱导晶状体上皮细胞TTase表达上调、活性增强,同时伴GSH的含量下降,GSSG含量上升。

Objective To observe the effects of high glucose on thioltransferase （TTase） expression in human lens epithelial cells. Methods Human lens epithelial cells B3（HLEC B3） were cultured in DMEM containing 15% fetal bovine serum and ex- posed to 5.5 mmol · L ^- 1 glucose （ control group ）, 15.5 mmol · L ^- 1,25.5 mmol · L^ -1, 35.5 mmol · L- J high glucose groups and mannitol control group. After 24 hours, incu- bation cells were gathered and TTase expression was examined by Western blotting. Cells were then gathered at 0,2 hours, 8 hours, 16 hours and 24 hours after incubation with 35.5 mmol · L^-1 high glucose, respectively. TTase expression and its activity were examined and the levels of T-GSH,GSH and GSSG of HLEC B3 were measured. GSSG! T-GSH ratio was calculated. Results With the raise of the concentration of high glu- cose, the expression of TYase increased gradually and the maximum expression ap- peared in 35.5 mmol · L^-1 group,almost 2.3 times to the control group（P 〈0.01 ）. No changes was observed in the mannitol control group （ P 〉 0. 05 ）. The expression of TTase increased after treated with high glucose of 35.5 mmol · L^-1 for 2 hours and reached the top for 8 hours,nearly 2.0 times to control group（P 〈 0.01 ） and then de- creased gradually. Similarly, TTase activity in 35.5 mmol· L^-1 group increased from 2 hours, reached at 8 hours, nearly 1.8 times to control group （ P 〈 0.01 ）, then decreased gradually. The level of GSH decreased after treated with 35.5 mmol · L^-1 glucose and was significantly lower at 16 hours than control group（P 〈 0.05 ）. The level of GSSG and the ratio of GSSG/T-GSH increased significantly after 2 hours incubation with 35. 5 mmol · L^-1 glucose, nearly 3.5 times （P 〈 0.001 ） and 3.3 times （P 〈 0.001 ） to the con- trol group, respectively, and continued to elevate with time. Conclusion The expression and activity of TTase in HLE cells increase after treatment with high glucose, in which the GSH level decrease and GSSG level increase.