金雀异黄素对过氧化氢损伤的人晶状体上皮细胞的作用及相关机制研究
发布时间：2018-07-17 01:01
【摘要】：研究目的 本课题拟通过研究中药植物雌激素金雀异黄素(genistein, Gen)对过氧化氢(H202)损伤的人晶状体上皮细胞(human lens epithelia cell, HLEC)的作用,从氧化损伤和细胞凋亡的角度探讨金雀异黄素防治白内障的细胞和分子机制,为寻求防治白内障的确切有效的药物提供科学的实验依据。 研究方法 本研究采用Gen与人晶状体上皮细胞(HLEC)共同孵育,以雌二醇(β-Estradiol,E2)作为阳性对照,再与H202作用后,进行下列研究： 一、采用四甲基偶氮唑蓝法(methyl thiazolyl tetrazolium, MTT)检测不同浓度Gen对H202损伤的HLEC活性的影响。 二、Gen对H202损伤的HLEC氧化损伤的影响：采用化学比色法检测HLEC内超氧化物歧化酶(superoxide dismutase, SOD)、过氧化氢酶(catalase, CAT)活性的变化,谷胱甘肽(glutathione, GSH)、丙二醛(malondialdehyde, MDA)的含量变化,探讨Gen的抗氧化作用及酶学机制。 三、Gen对H2O2损伤的HLEC凋亡的影响： 1、采用透射电子显微镜观察Gen对HLEC凋亡形态的影响。 2、采用流式细胞仪(flow cytometer, FCM)检测HLEC凋亡率,探讨Gen对HLEC凋亡率的影响。 3、采用FCM检测HLEC的线粒体跨膜电位(mitochondria membrane potential,ΔΨM)的变化,从线粒体依赖的凋亡通路探讨Gen影响HLEC凋亡的机制。 研究结果 一、MTT法检测显示,经H202处理的HLEC吸光度值(absorbance, A)降低；E2和Gen作用的HLEC吸光度值均较H202组显著升高。 二、Gen对H2O2引起的HLEC氧化损伤的影响： 1、H202组HLEC内SOD、CAT活性、GSH含量与正常组相比均显著降低(P0.01)；E2组与H2O2组相比,SOD、CAT活性均显著升高(P0.01),GSH含量也明显增加(P0.05)；Gen组与H202组相比,SOD活性显著升高(P0.01),CAT活性与GSH含量也明显升高(P0.05)。 2、H202组HLEC内MDA含量比正常组显著升高(P0.01)；E2组MDA含量比H202组明显降低(P0.05)；Gen组MDA含量与H202组相比显著降低(P0.01)。 三、Gen对H202所致的HLEC凋亡的影响： 1、电子显微镜观察凋亡形态的结果显示：H202组可见HLEC核染色质边集、固缩等细胞凋亡的改变；亦可见细胞整体结构破坏,胞浆内容物溶解消失,胞核碎裂等细胞坏死的改变。E2、Gen组与H202组比较,细胞凋亡的形态改变明显减轻、未出现坏死。 2、FCM检测显示,H202组凋亡率与正常组相比显著升高；E2组和Gen组凋亡率与H202组相比显著下降(P0.01)。 3、FCM检测显示,H202组线粒体跨膜电位与正常组相比显著下降；E2组和Gen组的线粒体跨膜电位与H202组相比显著升高(P0.01)。 研究结论 一、H202可引起HLEC发生氧化损伤。E2可减轻H202引起的HLEC氧化损伤,Gen亦可减轻H2O2引起的HLEC氧化损伤,表现出类雌激素样作用。 二、Gen减轻H2O2引起的HLEC氧化损伤的机制可能是通过提高HLEC内抗氧化酶SOD和CAT活性、增加非酶性抗氧化物GSH含量、降低膜脂质过氧化物MDA的含量实现的。 三、H2O2可引起HLEC发生凋亡。E2可减轻H202引起的HLEC凋亡,Gen亦可减轻H202引起的HLEC凋亡,表现出类雌激素样作用。 四、Gen减轻H2O2引起的HLEC凋亡的机制可能是通过降低细胞凋亡率、升高线粒体跨膜电位实现的。 五、上述结论提示,Gen作为一种植物雌激素,可能是发挥了类雌激素作用,通过提高H202损伤的HLEC的抗氧化能力、减轻其凋亡程度,对HLEC体现了保护作用。
[Abstract]:Purpose of study
This study intends to study the effect of genistein ( Gen ) on human lens epithelial cell ( HLEC ) injured by hydrogen peroxide ( H202 ) , and to explore the cellular and molecular mechanism of genistein in the prevention and treatment of cataract from oxidative damage and apoptosis .
Research Methods
In this study , Gen was incubated with human lens epithelial cells ( HLEC ) , and estradiol ( 尾 - carotene , E2 ) was used as positive control . After interacting with H202 , the following studies were carried out :
1 . The effect of different concentrations of Gen on HLEC activity was determined by MTT assay .
The effect of Gen on oxidative damage of HLEC was studied by chemical colorimetry . The changes of superoxide dismutase ( SOD ) , catalase ( catalase , CAT ) activity , glutathione ( GSH ) and malondialdehyde ( MDA ) in HLEC were detected by chemical colorimetry .
III . Effect of Gen on HLEC apoptosis induced by H2O2 :
1 . The effect of Gen on the apoptosis of HLEC was observed by transmission electron microscope .
2 . The apoptosis rate of HLEC was detected by flow cytometry ( FCM ) , and the effect of Gen on the apoptosis rate of HLEC was investigated .
3 . FCM was used to detect the changes of mitochondrial membrane potential ( 螖PSI M ) of HLEC , and the mechanism of Gen effect on HLEC apoptosis was discussed from mitochondria - dependent apoptotic pathways .
Results of the study
1 . The absorbance ( A ) of HLEC treated by H202 was reduced by MTT assay .
The HLEC absorbance values of E2 and Gen were significantly higher than those in H202 group .
II . Effect of Gen on HLEC Oxidation Damage Induced by H2O2 :
The activities of SOD , CAT and GSH in HLEC group were significantly lower than those in normal group ( P0.01 ) .
Compared with H2O2 group , the activity of SOD and CAT increased significantly ( P0.01 ) , GSH content increased significantly ( P0.05 ) .
Compared with group H202 , the activity of SOD increased significantly ( P0.01 ) , CAT activity and GSH content increased significantly ( P0.05 ) .
2 . The content of MDA in HLEC group was significantly higher than that in normal group ( P0.01 ) .
The content of MDA in the E2 group was significantly lower than that in H202 group ( P0.05 ) .
The content of MDA in Gen group was significantly lower than that of H202 group ( P0.01 ) .
III . Effect of Gen on HLEC apoptosis due to H202 :
1 . The results of electron microscopic observation of apoptotic morphology showed that HLEC could be seen in H202 group .
The changes of cell necrosis , such as the destruction of the whole cell structure , the disappearance of the contents of the cytoplasm , the nucleus fragmentation and the like were also observed .
2 . FCM showed that the apoptosis rate of H202 group was significantly higher than that in normal group .
Compared with H202 group , the apoptosis rate of E2 group and Gen group decreased significantly ( P0.01 ) .
3 . FCM analysis showed that the mitochondrial transmembrane potential of H202 group was significantly decreased compared with the normal group .
The mitochondrial transmembrane potential of the E2 group and the Gen group was significantly higher than that in the H202 group ( P0.01 ) .
Conclusions of the study
In addition , HLEC can reduce HLEC ' s oxidative damage caused by HLEC . Gen can also alleviate HLEC ' s oxidative damage caused by H2O2 , which shows the effect of estrogen - like effect .
2 . Gen alleviate HLEC ' s oxidative damage induced by H2O2 may be achieved by increasing the activity of SOD and CAT in HLEC , increasing the GSH content of non - enzymatic antioxidant , and lowering the content of MDA in membrane lipid peroxide .
3 . H2O2 can induce apoptosis of HLEC . E2 can alleviate HLEC apoptosis induced by H202 , and Gen can reduce HLEC apoptosis induced by H202 , and show the effect of estrogen - like sample .
4 . Gen alleviate HLEC apoptosis induced by H2O2 may be achieved by reducing cell apoptosis rate and increasing mitochondrial transmembrane potential .
5 . The above conclusion suggests that Gen as a phytoestrogen may play an important role in promoting the oxidation resistance of HLEC and reducing its degree of apoptosis by increasing HLEC ' s oxidation resistance , which is a protective effect on HLEC .
【学位授予单位】：福建中医药大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R285;R776.1
本文编号：2128329
[Abstract]:Purpose of study
This study intends to study the effect of genistein ( Gen ) on human lens epithelial cell ( HLEC ) injured by hydrogen peroxide ( H202 ) , and to explore the cellular and molecular mechanism of genistein in the prevention and treatment of cataract from oxidative damage and apoptosis .
Research Methods
In this study , Gen was incubated with human lens epithelial cells ( HLEC ) , and estradiol ( 尾 - carotene , E2 ) was used as positive control . After interacting with H202 , the following studies were carried out :
1 . The effect of different concentrations of Gen on HLEC activity was determined by MTT assay .
The effect of Gen on oxidative damage of HLEC was studied by chemical colorimetry . The changes of superoxide dismutase ( SOD ) , catalase ( catalase , CAT ) activity , glutathione ( GSH ) and malondialdehyde ( MDA ) in HLEC were detected by chemical colorimetry .
III . Effect of Gen on HLEC apoptosis induced by H2O2 :
1 . The effect of Gen on the apoptosis of HLEC was observed by transmission electron microscope .
2 . The apoptosis rate of HLEC was detected by flow cytometry ( FCM ) , and the effect of Gen on the apoptosis rate of HLEC was investigated .
3 . FCM was used to detect the changes of mitochondrial membrane potential ( 螖PSI M ) of HLEC , and the mechanism of Gen effect on HLEC apoptosis was discussed from mitochondria - dependent apoptotic pathways .
Results of the study
1 . The absorbance ( A ) of HLEC treated by H202 was reduced by MTT assay .
The HLEC absorbance values of E2 and Gen were significantly higher than those in H202 group .
II . Effect of Gen on HLEC Oxidation Damage Induced by H2O2 :
The activities of SOD , CAT and GSH in HLEC group were significantly lower than those in normal group ( P0.01 ) .
Compared with H2O2 group , the activity of SOD and CAT increased significantly ( P0.01 ) , GSH content increased significantly ( P0.05 ) .
Compared with group H202 , the activity of SOD increased significantly ( P0.01 ) , CAT activity and GSH content increased significantly ( P0.05 ) .
2 . The content of MDA in HLEC group was significantly higher than that in normal group ( P0.01 ) .
The content of MDA in the E2 group was significantly lower than that in H202 group ( P0.05 ) .
The content of MDA in Gen group was significantly lower than that of H202 group ( P0.01 ) .
III . Effect of Gen on HLEC apoptosis due to H202 :
1 . The results of electron microscopic observation of apoptotic morphology showed that HLEC could be seen in H202 group .
The changes of cell necrosis , such as the destruction of the whole cell structure , the disappearance of the contents of the cytoplasm , the nucleus fragmentation and the like were also observed .
2 . FCM showed that the apoptosis rate of H202 group was significantly higher than that in normal group .
Compared with H202 group , the apoptosis rate of E2 group and Gen group decreased significantly ( P0.01 ) .
3 . FCM analysis showed that the mitochondrial transmembrane potential of H202 group was significantly decreased compared with the normal group .
The mitochondrial transmembrane potential of the E2 group and the Gen group was significantly higher than that in the H202 group ( P0.01 ) .
Conclusions of the study
In addition , HLEC can reduce HLEC ' s oxidative damage caused by HLEC . Gen can also alleviate HLEC ' s oxidative damage caused by H2O2 , which shows the effect of estrogen - like effect .
2 . Gen alleviate HLEC ' s oxidative damage induced by H2O2 may be achieved by increasing the activity of SOD and CAT in HLEC , increasing the GSH content of non - enzymatic antioxidant , and lowering the content of MDA in membrane lipid peroxide .
3 . H2O2 can induce apoptosis of HLEC . E2 can alleviate HLEC apoptosis induced by H202 , and Gen can reduce HLEC apoptosis induced by H202 , and show the effect of estrogen - like sample .
4 . Gen alleviate HLEC apoptosis induced by H2O2 may be achieved by reducing cell apoptosis rate and increasing mitochondrial transmembrane potential .
5 . The above conclusion suggests that Gen as a phytoestrogen may play an important role in promoting the oxidation resistance of HLEC and reducing its degree of apoptosis by increasing HLEC ' s oxidation resistance , which is a protective effect on HLEC .
【学位授予单位】：福建中医药大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R285;R776.1
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