重组水蛭素Ⅲ对半乳糖损伤的人晶状体上皮细胞凋亡相关基因表达的影响  

Effects of recombinant hirudin variant HI on expression of apoptosis-related genes during galactose-mediated human lens epithelial cells damage

作　　者：欧瑜[1] 袁志军[1] 耿培[1] 吴梧桐[1] 

机构地区：[1]中国药科大学生命科学与技术学院,南京210009

出　　处：《中华实验眼科杂志》2011年第7期581-584,共4页Chinese Journal Of Experimental Ophthalmology

基　　金：教育部科学技术研究重点项目（S092006）

摘　　要：背景重组水蛭素Ⅲ（rHV3）对半乳糖损伤的人晶状体上皮细胞（LECs）有较好的保护作用，但其分子机制尚不清楚。目的探讨rHV3对半乳糖损伤的人LECs凋亡相关基因表达的影响。方法用本课题组构建的表达重组水蛭素工程菌，参照Markwardt的凝血酶滴定方法进行水蛭素生物学活性的测定。将培养的人LECs分为3组，培养于含质量分数10％胎牛血清（FBS）的D／F12培养液作为正常组、用含125×10^-3mol／LD一半乳糖的D／F12培养液建立人LECs株SRAOI／04损伤模型为模型组，用2000U／ml（商品单位）rHV3加入部分损伤模型培养液中进行干预作为rHV3组。采用逆转录聚合酶链反应（RT—PCR）法检测各组人LECs内凋亡相关基因的表达，包括醛糖还原酶（AR）、bcl2、bax、p53基因mRNA的表达水平，各目的基因表达丰度用目的基因的吸光度（A）值与3一磷酸甘油醛脱氢酶（GAPDH）A值的比值表达。结果与正常组比较，模型组人LECs中ARmRNA／GAPDHmRNA、baxmRNA／GAPDHmRNA和p53mRNA／GAPDHmRNA值明显高于对照组，差异均有统计学意义（t=3．90E一06、t=8．44E一04、t=5．15E一08，P〈0．叭），而水蛭素干预组人LECs中ARmRNA／GAPDHmRNA、baxmRNA／GAPDHmRNA和p53mRNA／GAPDHmRNA值明显低于模型组，差异均有统计学意义（t=5．90E一06、t=1．51E．04、t=3．42E一06，P〈0．01）。与正常组比较，模型组人LECs中的bcl2mRNA／GAPDHmRNA值明显降低，差异有统计学意义（t=1．86E一05，P〈O．01），与模型组相比，水蛭素干预后人LECs中的bcl2mRNA／GAPDHmRNA值显著提高，差异有统计学意义（t=8．56E一05，P〈0．01）。结论D一半乳糖导致人LECs损伤，rHV3可能通过抑制多元醇途径和线粒体介导的凋亡途径对人LECs起保护作用。Background Recombinant hirudin variant m （rHV3） can effectively prevent galactose-induced human lens epithelial ceils LECs injury,but little is known about the molecular mechanism of its action. Objective The present study was to investigate the effects of rHV3 on the expression of apoptosis-related genes in damaged LECs induced by galactose. Methods The rHV3 was extracted by our research group, and the biological activity of rHV3 was identified by titration of thrombase according to Markwardt＇ s method. Human LECs （SRAO1/04） were cultured using 125x10^-3 mol/L D-galactose＋ 10% FBS＋D/F12 medium to establish the damaged human LECs model, rHV3 was added into the medium of the damaged human LECs model. Human LECs were cultured in D/F12 medium containing 10% FBS as normal control. The expression of apoptosis-related genes, such as aldose reductase （AR） , bax,bel2 and p53 ,in LECs at the mRNA level was deteeted using RT-PCR. The abundance ratio of target genes waspresented with the absorbance （A） of gene mRNA/GAPDH mRNA. Results Compared to the normal control group ,the A values of AR mRNA/GAPDH mRNA,bax mRNA/GAPDH mRNA and p53 mRNA/GAPDH mRNA were significantly elevated in model group （t = 3.90E-06,t = 8.44E-04 ,t = 5. 15E-08 ,P〈0.01 ）. However, in the rHV3- treated group, the A values of AR mRNA/GAPDH mRNA, bax mRNA/GAPDH mRNA and p53 mRNA/GAPDH mRNA were lower than those of model group （t=5.90E-06,t= 1.51E-04,t=3.42E-06,P〈0.01 ）. The bcl2 mRNA/ GAPDH mRNA was markedly downregulated in the model group when compared with the normal control group （t = 1.86E-05 ,P〈0.01 ） ;while after rHV3 addition,bcl2 mRNA/GAPDH mRNA increased in comparison with the model group （t= 8.56E-05 ,P〈0.01 ）. Conclusion 125×10^-3 mol/L D-galactose induces the damage and apoptosis of human LECs. rHV3 likely plays a protective function on D-galactose-induced damage of human LECs by inhibiting the polyol pathway and mitochondria-mediated pathway.

关 键 词：人晶状体上皮细胞 重组水蛭素Ⅲ 半乳糖 凋亡 基因 

分 类 号：R776.1[医药卫生—眼科]