泪腺上皮细胞凋亡与丙酸睾酮的抑制效应

Inhibitory effects of testosterone propionate on the apoptosis of lacrimal gland epithelial cells

目的:分析丙酸睾酮抑制泪腺上皮细胞凋亡的机制,为干眼症的性激素防治提供科学的理论依据。方法:实验于2003-09/2004-01在重庆医科大学附属第一医院中心实验室完成。①实验材料:二三月龄新西兰健康白色家兔30只,无眼疾,雌性,体质量1.5～2.5kg。丙酸睾酮(产品批号:020902,上海通用药业股份有限公司产品),用DMSO配成储存液,-70℃保存。分析纯过氧化氢购自北方同正生物技术发展有限公司。②实验方法:经耳缘静脉注射空气处死家兔,按常规无菌操作行泪腺摘除术,酶消化法分离兔泪腺上皮细胞。取二三代培养的泪腺上皮细胞,按约1×108L-1密度接种于预置小玻片的24孔板内,培养4d后,加入浓度为1×10-8,1×10-7,1×10-6,1×10-5mol/L丙酸睾酮,继续培养24h,另以未加丙酸睾酮的培养液培养的同期传代的泪腺上皮细胞作空白对照。培养24h后,再分别加入终浓度为100μmol/LH2O2于不同浓度丙酸睾酮组继续培养60min,另以未加丙酸睾酮的DMEM/F12培养液培养的同期传代的泪腺上皮细胞作对照。③实验评估:应用TUNEL法检测泪腺上皮细胞凋亡,采用免疫细胞化学法检测泪腺上皮细胞Bax和Bcl-2表达。结果:①丙酸睾酮对过氧化氢诱导的泪腺上皮细胞凋亡的影响:与单纯加入100μmol/LH2O2组比较,加入不同浓度丙酸睾酮组泪腺上皮细胞凋亡率均明显下降且丙酸睾酮的抗凋亡作用呈现一定范围内的剂量依赖性[(15.15±1.05)%,(13.94±0.73)%,(12.51±0.91)%,(10.63±0.78)%,(8.19±0.85)%,P<0.01]。②免疫细胞化学法检测Bcl-2和Bax蛋白表达:与单纯加100μmol/LH2O2组相比较,同时加入1×10-8,1×10-7,1×10-6,1×10-5mol/L丙酸睾酮各组均可使泪腺上皮细胞Bcl-2的表达明显增强和Bax的表达明显减弱,且与丙酸睾酮的剂量呈正相关(Bcl-2:0.92±0.10,1.01±0.08,1.12±0.06,1.24±0.12,1.37±0.11;Bax:2.28±0.09,2.17±0.11,2.06±0.07,1.91±0.13,1.77±0.10,P<0.01)。结论:丙酸睾酮抑制泪腺上皮细胞凋亡的机制可能与凋亡调节相关基因Bcl-2表达上调和Bax表达下调有关。

AIM： To analyze the inhibitive effect of testosterone propionate on the apoptosis of lacrimal gland epithelial cells in order to provide scientific theoretical evidences hormone treating dry eye syndrome. METHODS： The experiment was conducted in the Central Laboratory of First Affiliated Hospital, Chongqing Medical University between September 2003 and January 2004. ①Totally 30 healthy female white New Zealand rabbits of 2-3 months without eye illness were used which weighed 1.5-2.5 kg. Testosterone propionate （Iot number 02 0902, Shanghai Tongyong Pharmaceutical Co., Ltd.） was prepared with DMSO as stock liquor and kept at -70 %. Analytical pure hydrogen peroxide was purchased from Beifang Tongzheng Biotechnology Development Co., Ltd. ② The rabbits were sacrificed by aeroembolism in entotic vein and took the lacrimal gland without germ conventionally. The isolation of rabbit lacrimal gland epithelial cells was adopted by enzyme digesting. 2-3 generation lacrimal gland epithelial cells were cultured in 24-well plate that contained 1 ×10^8 L^-1 for 4 days. Various concentrations of testosterone propionate （ 1 ×10^-8, 1 ×10^-7, 1 ×10^-6, 1 ×10^-5 mol/L ） were added and incubated for 24 hours, 2-3 generation lacrimal gland epithelial cells without testosterone propionate were used as blank control, and then 100 μmol/L H2O2 was added in various concentrations of testosterone propionate groups and incubated for 60 minutes, and 2-3 generation lacrimal gland epithelial cells cultured in DMEM/F12 culture fluid without testosterone propionate were applied as control. ③Cell apoptosis of lacrimal gland epithelial cells was detected by TUNEL technology. Expressions of Bcl-2 and Bax were detected by immunocytochemical method. RESULTS： ①Influence of testosterone propionate on apoptosis of lacrimal gland epithelial cells induced by hydrogen peroxide： As compared with the pure 100 μmol/L H2O2 group, apoptotic rates of lacrimal gland epithelial cells were obviously decreased in the various concentrations of testosterone propionate groups, and the anti-apoptosis of testosterone propionate showed dose-dependence to some degree [（15.15±1.05）%, （13.94±0.73）%, （12.51±0.91）%, （10.63±0.78）%, （8.19±0.85）%, P 〈 0.01]. ②Expressions of Bcl-2 and Bax detected by immunocytochemical method： As compared with the pure 100 μmol/L H2O2 group, expression of Bcl-2 strengthened significantly and expression of Bax weakened significantly after adding 1×10^-8, 1 ×10^-7, 1 ×10^-6, 1 ×10^-5 mol/L testosterone propionate, which showed positive correlation with the dose of testosterone propionate （Bcl-2：0.92±0.10,1.01±0.08, 1.12±0.06, 1.24±0.12, 1.37±0.11 ;Bax： 2.28±0.09, 2.17±0.11, 2.06±0.07, 1.91±0.13, 1.77±0.10, P〈 0.01）.