2型糖尿病患者与正常人泪液分泌及泪膜功能的比较

背景以往对糖尿病患者泪液分泌和泪膜功能研究的结果报道不一.近年来,应用泪液蛋白质评估泪膜的功能越来越受到关注.目的观察2型糖尿病患者和正常人泪液主要蛋白质含量及基础泪液分泌情况,探讨2型糖尿病患者泪液分泌及泪膜功能.设计病例-对照观察.单位解放军第三军医大学西南医院眼科.对象选择2001-12/2002-12第三军医大学西南医院眼科和内分泌科确诊的2型糖尿病患者50例(100眼).均无眼部手术史及激光治疗史、近期眼局部用药史、接触镜配戴史.其中增殖性糖尿病视网膜病变组25例(50眼);非增殖性糖尿病视网膜病变组25例(50眼).另外选取年龄性别相匹配正常健康体检者25例(50眼)作对照.3组年龄及性别构成比差异无显著性(χ2=0.024,0.321;P＞0.05),实验开始前均获参与者知情同意.方法[1]泪液的采集随机抽取两组糖尿病患者及对照者各10例(20眼).采用毛细吸管法在下泪河收集10 μL非刺激性泪液,-20℃冰箱中保存备用(＜1个月).[2]泪液总蛋白量的测定采用Lorry法测定泪液总蛋白浓度,用小牛血清白蛋白作为标准.[3]泪液主要蛋白质含量测定采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法,考马斯亮蓝染色,采用Bio-Rad图像分析系统,对分离出的蛋白质条带作定性、定量分析.[4]泪膜破裂时间测定用玻璃棒沾取20 g/L的荧光素钠滴入检查者结膜囊,嘱被检查者轻轻眨眼数次,自然睁眼平视前方.直至发现完整泪膜出现第1个破孔"黑洞"的时间,即泪膜破裂时间.[5]基础泪液分泌试验用Whatmann 41号滤纸,折5 mm置于下睑外1/3结膜处,5 min后取下滤纸,测定滤纸湿后长度.[6]孟加拉玫瑰红染色试验用玻璃棒沾取10 g/L孟加拉玫瑰红染料滴入结膜囊瞬目数分钟之后,在裂隙灯下用无赤光观察(评分标准为以睑裂部角结膜染色为+,染色累及下方球结膜为++,累及上方球结膜为+++),以其染红上皮细胞和黏蛋白间接诊断干眼病.主要观察指标[1]泪液总蛋白质浓度.[2]各种泪液主要蛋白质浓度.[3]泪膜破裂时间.[4]基础泪液分泌测定值.[5]孟加拉玫瑰红染色阳性率.结果糖尿病组50例(100眼)和对照组25例(50眼)均进入结果分析.[1]泪液总蛋白质浓度3组比较差异无显著性(P＞0.05).[2]各种泪液主要蛋白质浓度增殖性糖尿病视网膜病变组与对照组比较,溶菌酶、乳铁蛋白及泪液特异性前白蛋白明显降低[(0.94±0.21)比(1.33±0.31)g/L,(1.10±0.24)比(1.67±0.43)g/L,(0.98±0.22)比(1.49±0.32)g/L,P＜0.01],与非增殖性糖尿病视网膜病变组比较,乳铁蛋白和泪液特异性前白蛋白降低(P＜0.05).人血清白蛋白3组比较差异无显著性(P＞0.05).[3]泪膜破裂时间增殖性糖尿病视网膜病变组与非增殖性糖尿病视网膜病变组和对照组比较,泪膜破裂时间显著降低[(7.68±2.21)s比(9.92±2.37)和(10.80±2.23)s,P＜0.01].[4]基础泪液分泌测定值增殖性糖尿病视网膜病变组测定值显著小于非增殖性糖尿病视网膜病变组和对照组[(8.00±2.10)比(11.02±1.97)和(12.17±2.08)mm,P＜0.05].[5]孟加拉玫瑰红染色阳性率增殖性糖尿病视网膜病变组阳性率显著高于非增殖性糖尿病视网膜病变组和对照组(48%比24%和14%,P＜0.05,P＜0.01).结论本文结果提示2型糖尿病患者易发生泪液分泌和泪膜功能异常,尤其是增殖性糖尿病视网膜病变患者泪膜功能降低更加明显.采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法有助于发现糖尿病患者泪液蛋白质的变化.

Comparison of tear secretion and tear film function between patients with type 2 diabetes mellitus and normal people

BACKGROUND: There are various reports on studies of tear secretion and tear film function in patients with diabetes mellitus over the past. In recent years, tear proteins have drawn more and more attentions on evaluation of tear film function.OBJECTIVE: To observe the contents of main tear proteins and basal tear secretion of patients with type 2 diabetes mellitus and normal persons so as to probe into tear secretion and tear film function of patients with diabetes mellitus.DESIGN: Case-control observation was designed.SETTING: Department of Ophthalmology, Southwest Hospital, Third Military Medical University of Chinese PLA.PARTICIPANTS: Totally 50 cases (100 eyes) of type 2 diabetes mellitus were employed, which were diagnosed in Department of Ophthalmology and Department of Endocrinology, Southwest Hospital of Third Military Medical University of Chinese PLA from December 2001 to December 2002. They were free from ocular surgical and laser treatment, local medication in recent period and contact lens. Of those, there were 25 cases (50 eyes) in proliferating diabetic retinopathy group and 25 cases (50 eyes) in nonproliferating diabetic retinopathy group. In addition, 25 cases (50 eyes) of normal persons with matched age and sex were taken as the control group.There was no significant difference in age and sex among 3 groups (x2=0.024,0.321 ;P ＞ 0.05). All of the participants were in the know before the experiment.METHODS: [1] Tear collection: 10 cases (20 eyes) were randomized from two diabetic groups and the control successively. Capillary pipette method was used to collect non-irritative tear 10 μL from lower lacrimal punctum that was preserved in refrigerator at -20 ℃ (＜ 1 month). [2] Determination of total tear protein amount: Lorry method was used to determine the concentration of total tear protein, in which, calf serum albumin was taken as the criteria. [3] Determination of contents of main tear proteins: SDS-PAGE (sod.dodecyl sulfate-polyacrylamide gel electrophoresis) was used and Coomassie brilliant blue staining and Bio-Rad imaging analyzing system were applied for the analysis of isolated protein strips in quality and quantity. [4] Determination of rupture time of tear film: glass rod was used to get 20 g/L fluorescein sodium and drop in conjunctival sac. The examined person was required to blink gently for several times and open the eyes naturally and stare forward. That the first ruptured "black hole" was discovered on the complete tear film was taken as the rupture time of tear film. [5] Experiment of basal tear secretion: No.41 Whatmann filtering paper was used, folded in 5 mm, and placed at 1/3 of conjunctiva in the lower eyelid. Five minutes later, the paper was removed and length of it after wetting was measured. [6]Experiment with rose Bengal staining: glass rod was used to get 10 g/L rose Bengal and drop in conjunctival sac. After eyes blinking for several minutes, the observation was performed with green light filter under slit lamp (evaluation criteria: corneal conjunctiva of palpebral fissure stained+, stained to the inferior bulbar conjunctiva++,stained to the superior bulbar conjunctiva). Dry eye disease was diagnosed indirectly with red-stained epithelial cells and mucin.MAIN OUTCOME MEASURES: [1] Concentration of total tear protein. [2]Concentrations of various main tear proteins. [3] Rupture time of tear film. [4]Value of basal tear secretion. [5] Positive rate of rose Bengal staining.RESULTS: Totally 50 cases (100 eyes) in diabetes groups and 25 cases(50 eyes) in the control group all entered result analysis. [1] Concentration of total tear protein: there was no significant difference among 3 groups (P＞ 0.05). [2] Concentrations of various main tear proteins: the results of lysozyme, lactoferritin and tear specific prealbumin in proliferating diabetic retinopathy group were lower remarkably compared with the control[ (0.94±0.21)vs ( 1.33±0.31 )g/L , ( 1.10±0.24)vs ( 1.67±0.43 )g/L, (0.98±0.22) vs (1.49±0.32)g/L, P ＜ 0.01]. Compared with non-proliferating diabetic retinopathy group, the results of lactoferritin and tear specific prealbumin were lower (P ＜ 0.05). There was no significant difference in human serum albumin among 3 groups (P ＞ 0.05). [3] Rupture time of tear film:compared with non-proliferating diabetic retinopathy group and the control, the rupture time of tear film in proliferating diabetic retinopathy group was reduced significantly [(7.68±2.21)s vs (9.92±2.37)s and(10.80±2.23)s,(P ＜ 0.01 )]. [4] Value of basal tear secretion: the value in proliferating diabetic retinopathy group was less significantly than that in non-proliferating diabetic retinopathy group and the control [ (8.00±2.10)vs( 11.02± 1.97 )mm and ( 12.17±2.08 )mm, P ＜ 0.05]. [5] Positive rate of rose Bengal staining: the positive rate in proliferating.diabetic retinopathy group was higher significantly than non-proliferating diabetic retinopathy group and the control (48% vs 24% and 14%, P ＜ 0.05, P ＜ 0.01).CONCLUSION: It is suggested in the results of this paper that abnormal tear secretion and tear film function are apt to present in patients with type 2 diabetes mellitus and the declined tear film function is more remarkably in the patients with proliferating diabetic retinopathy specially. SDS-PAGE benefits the discovery of changes in tear proteins in diabetic patients.