不同浓度17β-雌二醇对离体培养雌性大鼠晶状体上皮细胞凋亡的影响

发布时间：2018-07-10 17:08

本文选题：雌二醇 + 晶状体上皮细胞　；参考：《泸州医学院》2010年硕士论文

【摘要】：目的：观察雌激素中活性最强的17β-雌二醇(17β-estradiol, 17β-E2)在不同浓度时对过氧化氢(hydrogen dioxide, H2O2)诱导的雌性大鼠晶状体上皮细胞凋亡的影响及其对凋亡相关蛋白Bcl-2和Bax表达的调控,探讨不同浓度雌激素对晶状体上皮细胞凋亡的作用及其机制,为合理应用雌激素防治白内障提供实验依据。方法：摘取健康成年雌性SD大鼠的透明晶状体48枚,并置于Medium 199培养液中培养。按培养液中添加的成分差异随机分为6组：17β-E2+H2O2组1、17β-E2+H2O2组2、17β-E2+H2O2组3、17β-E2+H2O2组4、H2O2组以及空白对照组。予以终浓度为300μmol/L的过氧化氢复制SD大鼠晶状体上皮细胞凋亡模型,分别加入不同终浓度的17β-雌二醇干预17β-E2+H2O2组1至4(干预浓度分别为：1×10-8mol/L, 1×10-7mol/L, 1×10-6mol/L, 1×10-5mol/L。于CO2细胞恒温培养箱中共同孵育24小时后观察并比较各组晶状体混浊情况；每组取2枚晶状体行石蜡包埋、切片、苏木素-伊红染色(hematoxylin and eosin stain, HE stain);撕取各组剩余晶状体(6枚/组)前囊及赤道部囊膜铺片,采用脱氧核糖核酸末端转移酶缺口标记原位细胞凋亡检测法(terminal deoxynucleotidyl transferasa(TdT)-medinted dUTP nick-end labeling, TUNEL法)检测晶状体上皮细胞凋亡,链霉菌抗生物素蛋白-过氧化物酶连接法(Streptavidin Peroxidase Conjugated Method, SP法)检测凋亡相关蛋白Bcl-2和Bax的表达并进行比较。统计学分析：SPSS13.0单因素方差分析,并选用LSD法及SNK法进行两两比较。结果：经24小时孵育,空白对照组晶状体完全透明,H2O2组及17β-E2+H2O2各组晶状体混浊,但17β-E2+H2O2各组晶状体混浊程度均低于H2O2组,差异具有统计学意义(P0.05)；TUNEL法检测细胞凋亡结果显示：终浓度为300μmol/L的过氧化氢能成功诱导晶状体上皮细胞凋亡,17β-E2+H2O2各组晶状体上皮细胞凋亡率均显著低于H2O2组,差异具有统计学意义(P0.05),且与17β-雌二醇的浓度呈负相关(均数图Means Plots)。免疫组化SP法检测Bcl-2及Bax阳性表达率结果显示：在空白对照组晶状体上皮细胞中,Bcl-2和Bax均有表达,且Bcl-2的表达远较Bax表达强,Bcl-2/Bax比值处于较高水平；过氧化氢可明显降低Bcl-2表达,提高Bax表达(P0.05),Bcl-2/Bax比值下降；与H2O2组比较,17β-雌二醇可明显提高Bcl-2表达,降低Bax表达(P0.05),Bcl-2/Bax比值升高,且在一定范围内与17β-雌二醇浓度呈正相关(均数图Means Plots)。结论：17β-雌二醇对过氧化氢诱导的雌性大鼠晶状体上皮细胞凋亡有抑制作用,此抑制作用在一定范围内与17β-雌二醇的浓度呈正相关。对凋亡相关蛋白Bcl-2和Bax表达的调控可能是17β-雌二醇抑制晶状体上皮细胞凋亡的分子机制之一。
[Abstract]:Aim: to investigate the effects of 17 尾 -estradiol (17 尾 -E2), the most active estrogen, on the apoptosis of female rat lens epithelial cells induced by hydrogen peroxide (hydrogen dioxide, H _ 2O _ 2) at different concentrations and the regulation of Bcl-2 and Bax expression. To investigate the effect of estrogen at different concentrations on lens epithelial cell apoptosis and its mechanism, and to provide experimental evidence for the rational use of estrogen in the prevention and treatment of cataract. Methods: 48 clear lenses of healthy adult female SD rats were obtained and cultured in medium 199 medium. According to the difference of the components added in the culture medium, they were randomly divided into 6 groups: 1 / 17 尾 -E2 H _ 2O _ 2 group (1 / 17 尾 -E _ 2) H _ 2O _ 2 group ~ (2) ~ (2) ~ (17) 尾 -E _ (2) H _ 2O _ (2) _ (3) H _ 2O _ 2 group (n = 4) and blank control group (n = 6). SD rat lens epithelial cell apoptosis model was induced by hydrogen peroxide at the final concentration of 300 渭 mol / L, and 17 尾 -estradiol at different final concentrations was added to treat 17 尾 -E2 H _ 2O _ 2 group 1 to 4 (1 脳 10 ~ (-8) mol / L, 1 脳 10 ~ (-7) mol / L, 1 脳 10 ~ (-6) mol 路L ~ (-1), 1 脳 10 ~ (-5) mol / L) respectively. After incubating in a CO _ 2 cell incubator for 24 hours, the lens opacity in each group was observed and compared. (hematoxylin and eosin stain, HE stain); stained with hematoxylin and eosin were used to tear the anterior capsule and equatorial capsule of the remaining lens (6 / group). Apoptosis of lens epithelial cells was detected by deoxyribonucleic acid terminal transferase Nick labeling (terminal deoxynucleotidyl transferasa (TDT) -meditated dUTP nick-end labeling (Tunel method). Streptavidin Peroxidase conjugated method (SP) was used to detect the expression of apoptosis-related proteins Bcl-2 and Bax. The single factor ANOVA was analyzed by: SPSS 13.0, and the LSD method and SNK method were used to carry out the comparison. Results: after 24 hours incubation, the lens opacity in the blank control group was completely transparent and transparent, but the degree of lens opacity in 17 尾 -E2 H 2O 2 group was lower than that in H 2O 2 group (P0.05). The results of Tunel assay showed that hydrogen peroxide with final concentration of 300 渭 mol / L could successfully induce apoptosis of lens epithelial cells. The apoptosis rate of lens epithelial cells in H _ 2O _ 2 group was significantly lower than that in H _ 2O _ 2 group. The difference was statistically significant (P0.05) and negatively correlated with the concentration of 17 尾 -estradiol (mean means lots). The expression of Bcl-2 and Bax in lens epithelial cells of blank control group was detected by immunohistochemical SP method. The expression of Bcl-2 and Bax was much higher than that of Bax, and hydrogen peroxide could significantly decrease the expression of Bcl-2. Compared with H2O2 group, 17 尾 -estradiol significantly increased Bcl-2 expression, decreased Bax expression (P0.05) and increased Bcl-2 / Bax ratio, and was positively correlated with 17 尾 -estradiol concentration in a certain range (mean figure means lots). Conclusion the effect of 1: 17 尾 -estradiol on the apoptosis of female rat lens epithelial cells induced by hydrogen peroxide is positively correlated with the concentration of 17 尾 -estradiol. The regulation of apoptosis-related proteins Bcl-2 and Bax may be one of the molecular mechanisms of 17 尾 -estradiol inhibiting the apoptosis of lens epithelial cells.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R776.1

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