發(fā)布時(shí)間：2018-07-17 01:01

【摘要】：研究目的 本課題擬通過研究中藥植物雌激素金雀異黃素(genistein, Gen)對(duì)過氧化氫(H202)損傷的人晶狀體上皮細(xì)胞(human lens epithelia cell, HLEC)的作用,從氧化損傷和細(xì)胞凋亡的角度探討金雀異黃素防治白內(nèi)障的細(xì)胞和分子機(jī)制,為尋求防治白內(nèi)障的確切有效的藥物提供科學(xué)的實(shí)驗(yàn)依據(jù)。 研究方法 本研究采用Gen與人晶狀體上皮細(xì)胞(HLEC)共同孵育,以雌二醇(β-Estradiol,E2)作為陽性對(duì)照,再與H202作用后,進(jìn)行下列研究： 一、采用四甲基偶氮唑藍(lán)法(methyl thiazolyl tetrazolium, MTT)檢測(cè)不同濃度Gen對(duì)H202損傷的HLEC活性的影響。 二、Gen對(duì)H202損傷的HLEC氧化損傷的影響：采用化學(xué)比色法檢測(cè)HLEC內(nèi)超氧化物歧化酶(superoxide dismutase, SOD)、過氧化氫酶(catalase, CAT)活性的變化,谷胱甘肽(glutathione, GSH)、丙二醛(malondialdehyde, MDA)的含量變化,探討Gen的抗氧化作用及酶學(xué)機(jī)制。 三、Gen對(duì)H2O2損傷的HLEC凋亡的影響： 1、采用透射電子顯微鏡觀察Gen對(duì)HLEC凋亡形態(tài)的影響。 2、采用流式細(xì)胞儀(flow cytometer, FCM)檢測(cè)HLEC凋亡率,探討Gen對(duì)HLEC凋亡率的影響。 3、采用FCM檢測(cè)HLEC的線粒體跨膜電位(mitochondria membrane potential,ΔΨM)的變化,從線粒體依賴的凋亡通路探討Gen影響HLEC凋亡的機(jī)制。 研究結(jié)果 一、MTT法檢測(cè)顯示,經(jīng)H202處理的HLEC吸光度值(absorbance, A)降低；E2和Gen作用的HLEC吸光度值均較H202組顯著升高。 二、Gen對(duì)H2O2引起的HLEC氧化損傷的影響： 1、H202組HLEC內(nèi)SOD、CAT活性、GSH含量與正常組相比均顯著降低(P0.01)；E2組與H2O2組相比,SOD、CAT活性均顯著升高(P0.01),GSH含量也明顯增加(P0.05)；Gen組與H202組相比,SOD活性顯著升高(P0.01),CAT活性與GSH含量也明顯升高(P0.05)。 2、H202組HLEC內(nèi)MDA含量比正常組顯著升高(P0.01)；E2組MDA含量比H202組明顯降低(P0.05)；Gen組MDA含量與H202組相比顯著降低(P0.01)。 三、Gen對(duì)H202所致的HLEC凋亡的影響： 1、電子顯微鏡觀察凋亡形態(tài)的結(jié)果顯示：H202組可見HLEC核染色質(zhì)邊集、固縮等細(xì)胞凋亡的改變；亦可見細(xì)胞整體結(jié)構(gòu)破壞,胞漿內(nèi)容物溶解消失,胞核碎裂等細(xì)胞壞死的改變。E2、Gen組與H202組比較,細(xì)胞凋亡的形態(tài)改變明顯減輕、未出現(xiàn)壞死。 2、FCM檢測(cè)顯示,H202組凋亡率與正常組相比顯著升高；E2組和Gen組凋亡率與H202組相比顯著下降(P0.01)。 3、FCM檢測(cè)顯示,H202組線粒體跨膜電位與正常組相比顯著下降；E2組和Gen組的線粒體跨膜電位與H202組相比顯著升高(P0.01)。 研究結(jié)論 一、H202可引起HLEC發(fā)生氧化損傷。E2可減輕H202引起的HLEC氧化損傷,Gen亦可減輕H2O2引起的HLEC氧化損傷,表現(xiàn)出類雌激素樣作用。 二、Gen減輕H2O2引起的HLEC氧化損傷的機(jī)制可能是通過提高HLEC內(nèi)抗氧化酶SOD和CAT活性、增加非酶性抗氧化物GSH含量、降低膜脂質(zhì)過氧化物MDA的含量實(shí)現(xiàn)的。 三、H2O2可引起HLEC發(fā)生凋亡。E2可減輕H202引起的HLEC凋亡,Gen亦可減輕H202引起的HLEC凋亡,表現(xiàn)出類雌激素樣作用。 四、Gen減輕H2O2引起的HLEC凋亡的機(jī)制可能是通過降低細(xì)胞凋亡率、升高線粒體跨膜電位實(shí)現(xiàn)的。 五、上述結(jié)論提示,Gen作為一種植物雌激素,可能是發(fā)揮了類雌激素作用,通過提高H202損傷的HLEC的抗氧化能力、減輕其凋亡程度,對(duì)HLEC體現(xiàn)了保護(hù)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 .
【學(xué)位授予單位】：福建中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R285;R776.1

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