中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2011年
12期
1082-1086
,共5页
沙翔垠%罗春云%宋莉%樊飞红%贺小松%杜道兵
沙翔垠%囉春雲%宋莉%樊飛紅%賀小鬆%杜道兵
사상은%라춘운%송리%번비홍%하소송%두도병
干眼症%泪腺上皮细胞%凋亡%雌二醇%丙酸睾酮%过氧化氢
榦眼癥%淚腺上皮細胞%凋亡%雌二醇%丙痠睪酮%過氧化氫
간안증%루선상피세포%조망%자이순%병산고동%과양화경
Dry eye%Lacrimal gland cell%Apoptosis%Estradiol%Testosterone%H2O2
背景 性激素在干眼症的发病过程中起着重要的作用,尤其是对泪腺的功能起到非常重要的调控作用,但其对泪腺上皮细胞凋亡作用的机制尚不完全明确.目的 探讨雌二醇及丙酸睾酮对H2O2诱导的兔泪腺上皮细胞的凋亡作用.方法 分离2～3月龄雄性普通级新西兰兔的泪腺组织,用组织块培养法体外培养兔泪腺上皮细胞,用细胞角蛋白(CK)抗体对培养的细胞进行免疫细胞化学鉴定.取对数生长期的细胞,以5×104个/ml接种于96孔板培养44 h后,培养基中分别加入1×10-5、1×10-6、1×10-7、1×10-8 mol/L的雌二醇或丙酸睾酮培养24 h,再加入1×10-4 mol/L H2O2培养1h诱导细胞凋亡,仅用H2O2作用的细胞作为凋亡对照组,常规培养的细胞作为空白对照组.MTT比色法检测各组细胞570 nm处泪腺上皮细胞活性的吸光度(A570)值,流式细胞仪Annexin V/PI双染法检测各组细胞的凋亡情况.结果 培养的细胞呈不规则多边形,其中80％以上的细胞对CK呈阳性反应.MTT比色法检测表明,与空白对照组比较,凋亡对照组、不同浓度雌二醇组和丙酸睾酮组的A570值均明显降低,差异均有统计学意义(P＜0.01),1×10-5、1×10-6、1×10-7mol/L雌二醇组和1×10-6 mol/L丙酸睾酮组的A570值均明显高于凋亡对照组,差异均有统计学意义(P＜0.01).与相应浓度丙酸睾酮组相比,雌二醇组A570值均明显增高,差异均有统计学意义(P＜0.01).与凋亡对照组比较,丙酸睾酮组和雌二醇组早期及晚期细胞凋亡率均明显下降,差异均有统计学意义(P＜0.01,P＜0.05)；与丙酸睾酮组相比,雌二醇组早期及晚期的细胞凋亡率明显下降,差异均有统计学意义( P＜0.01,P＜0.05).结论 雌二醇及丙酸睾酮可抑制H2O2诱导的泪腺上皮细胞的凋亡,雌二醇的抑制作用强于丙酸睾酮.雌二醇对泪腺上皮细胞凋亡的抑制作用呈一定的剂量依赖性.
揹景 性激素在榦眼癥的髮病過程中起著重要的作用,尤其是對淚腺的功能起到非常重要的調控作用,但其對淚腺上皮細胞凋亡作用的機製尚不完全明確.目的 探討雌二醇及丙痠睪酮對H2O2誘導的兔淚腺上皮細胞的凋亡作用.方法 分離2～3月齡雄性普通級新西蘭兔的淚腺組織,用組織塊培養法體外培養兔淚腺上皮細胞,用細胞角蛋白(CK)抗體對培養的細胞進行免疫細胞化學鑒定.取對數生長期的細胞,以5×104箇/ml接種于96孔闆培養44 h後,培養基中分彆加入1×10-5、1×10-6、1×10-7、1×10-8 mol/L的雌二醇或丙痠睪酮培養24 h,再加入1×10-4 mol/L H2O2培養1h誘導細胞凋亡,僅用H2O2作用的細胞作為凋亡對照組,常規培養的細胞作為空白對照組.MTT比色法檢測各組細胞570 nm處淚腺上皮細胞活性的吸光度(A570)值,流式細胞儀Annexin V/PI雙染法檢測各組細胞的凋亡情況.結果 培養的細胞呈不規則多邊形,其中80％以上的細胞對CK呈暘性反應.MTT比色法檢測錶明,與空白對照組比較,凋亡對照組、不同濃度雌二醇組和丙痠睪酮組的A570值均明顯降低,差異均有統計學意義(P＜0.01),1×10-5、1×10-6、1×10-7mol/L雌二醇組和1×10-6 mol/L丙痠睪酮組的A570值均明顯高于凋亡對照組,差異均有統計學意義(P＜0.01).與相應濃度丙痠睪酮組相比,雌二醇組A570值均明顯增高,差異均有統計學意義(P＜0.01).與凋亡對照組比較,丙痠睪酮組和雌二醇組早期及晚期細胞凋亡率均明顯下降,差異均有統計學意義(P＜0.01,P＜0.05)；與丙痠睪酮組相比,雌二醇組早期及晚期的細胞凋亡率明顯下降,差異均有統計學意義( P＜0.01,P＜0.05).結論 雌二醇及丙痠睪酮可抑製H2O2誘導的淚腺上皮細胞的凋亡,雌二醇的抑製作用彊于丙痠睪酮.雌二醇對淚腺上皮細胞凋亡的抑製作用呈一定的劑量依賴性.
배경 성격소재간안증적발병과정중기착중요적작용,우기시대루선적공능기도비상중요적조공작용,단기대루선상피세포조망작용적궤제상불완전명학.목적 탐토자이순급병산고동대H2O2유도적토루선상피세포적조망작용.방법 분리2～3월령웅성보통급신서란토적루선조직,용조직괴배양법체외배양토루선상피세포,용세포각단백(CK)항체대배양적세포진행면역세포화학감정.취대수생장기적세포,이5×104개/ml접충우96공판배양44 h후,배양기중분별가입1×10-5、1×10-6、1×10-7、1×10-8 mol/L적자이순혹병산고동배양24 h,재가입1×10-4 mol/L H2O2배양1h유도세포조망,부용H2O2작용적세포작위조망대조조,상규배양적세포작위공백대조조.MTT비색법검측각조세포570 nm처루선상피세포활성적흡광도(A570)치,류식세포의Annexin V/PI쌍염법검측각조세포적조망정황.결과 배양적세포정불규칙다변형,기중80％이상적세포대CK정양성반응.MTT비색법검측표명,여공백대조조비교,조망대조조、불동농도자이순조화병산고동조적A570치균명현강저,차이균유통계학의의(P＜0.01),1×10-5、1×10-6、1×10-7mol/L자이순조화1×10-6 mol/L병산고동조적A570치균명현고우조망대조조,차이균유통계학의의(P＜0.01).여상응농도병산고동조상비,자이순조A570치균명현증고,차이균유통계학의의(P＜0.01).여조망대조조비교,병산고동조화자이순조조기급만기세포조망솔균명현하강,차이균유통계학의의(P＜0.01,P＜0.05)；여병산고동조상비,자이순조조기급만기적세포조망솔명현하강,차이균유통계학의의( P＜0.01,P＜0.05).결론 자이순급병산고동가억제H2O2유도적루선상피세포적조망,자이순적억제작용강우병산고동.자이순대루선상피세포조망적억제작용정일정적제량의뢰성.
Background The sex hormones plays an important role in the incidence of dry eye,especially for the regulation of function.However,the effects of sex hormones on lacrimal gland epithelial cells are below understand.Objective This study was to investgate the effects of estradiol and testosterone on the apoptosis of lacrimal gland cells induced by H2O2.Methods The lacrimal gland tissue was obtained from 2- or 3-month-old clean male New Zealand rabbits and the lacrimal gland epithelial cells were cultured in vitro using esplant culture method.The cells were identified by pan cytokeratin antibodies with immunocytochemistry.lacrimal gland epithelial cells were incubated in the 96 well plate at the density of 5 × l04 cells/ml for 44 hours.Estradiol or testosterone with the concentrations of 1 × 10-5,1 × 10-6,1 × 10-7,1 × 10-8 mol/L were added into the medium for 24 hours respectively and 1× 10-4 mol/L H2O2 treated the cells for 1 hour to induce the apoptosis in experimental groups.The cells treated by only 1 × 10-4 mol/L H2O2 were used as apoptotic control group,and the cells cultured by regular method were used as blank control group.The cell viability in different groups was detected using MTT at 570 nm ( A570 ),and the apoptotic rates of the cells were assayed using Annexin V/PI double staining.This use and maintain of experimental animals followed the Regulation for the Administration of Affairs Concerning Experimental Animals by State Science and Technology Commission.Results The cultured cells showed the irregular polygon in shape,and about 80％ cells was positive response for cytokeratin.MTT assay showed that the lower A570 values were detected in the H2O2-induced group,various concentrations of estradiol or testosterone groups compared with blank control group (P＜0.01 ).The A570 values in 1 × 10-5,1 × 10-6,1 × 10-7 mol/L estradiol groups or 1 × 10-6 mol/L testosterone group were significantly higher than ones of H2 O2-induced group (P＜0.01 ).Compared with corresponding concentrations of testosterone groups,the A570values in various concentrations of estradiol groups were elevated( P＜0.01 ).The apoptosis rates at the early and later phase were significantly declined in both estradiol group and testosterone group in comparison with H2 O2-induced group (P ＜ 0.01,P＜ 0.05 ),and those in estradiol group were lower than the testosterone group( P＜0.01,P＜0.05 ).Conclusions Estradiol and testosterone suppress the apoptosis of lacrimal gland cells induced by H2O2,and the stronger effect is found in estrogen.The inhibition of estrogen on lacrimal gland cell apoptosis show a dose-dependent manner to some extent.
