糖尿病小鼠睑板腺功能障碍及其炎性因子和脂类代谢因子表达变化

Meibomian gland dysfunction and expressions of inflammatory factors and lipid metabolic factors in diabetic mice

目的观察糖尿病模型小鼠睑板腺形态和功能及睑板腺组织中炎性因子和脂类代谢因子的表达变化。方法采用随机数字表法将清洁级8周龄雄性C57BL/6小鼠50只分为正常对照组20只和糖尿病模型组30只。采用腹腔内注射10 mg/ml链脲佐菌素法制备糖尿病模型,鼠尾静脉血糖≥16.7 mmol/L视为造模成功,每周监测血糖,对比2个组小鼠体质量,每4周2个组任意选取10只小鼠行角膜荧光素钠染色评估角膜上皮完整性,于造模后8周和16周每组任意选取5只小鼠取睑板腺组织,行苏木精-伊红染色观察组织形态学变化;造模后16周每组任意选取5只小鼠对睑板腺组织行油红O染色对比观察睑酯分布情况;造模后16周,采用实时荧光定量PCR法检测睑板腺组织中肿瘤坏死因子(TNF)-α、色素上皮衍生因子(PEDF)、过氧化物酶体增殖物激活受体γ(PPARγ)和脂类分化相关蛋白(ADFP)mRNA的相对表达量。结果糖尿病模型组小鼠成模率为100%,饲养过程中存活率为83.3%(25/30)。糖尿病模型组小鼠造模后8周和16周体质量均较同期正常对照组明显降低,血糖较同期正常对照组升高,差异均有统计学意义(均P<0.05)。糖尿病模型组不同时间点角膜荧光素钠染色评分值比较差异有统计学意义(F=27.155,P<0.05)。造模后16周糖尿病模型组睑板腺导管管壁变薄,管腔扩大,腺泡膨胀,睑板腺大部分腺泡内油红O着染。造模后16周,糖尿病模型组睑板腺组织中TNF-α和PPARγ mRNA相对表达量分别为3.33±0.91和1.55±0.25,明显高于正常对照组的1.00±0.16和1.00±0.27,PEDF mRNA相对表达量为0.42±0.08,明显低于正常对照组的1.00±0.34,差异均有统计学意义(均P<0.05);2个组间ADFP mRNA相对表达量比较,差异无统计学意义(t=0.943,P=0.38)。结论 TNF-α、PEDF和PPARγ可能参与糖尿病诱导睑板腺功能障碍的发生。

Objective To explore the changes in morphology and function of meibomian gland and the expressions of inflammatory factors and lipid metabolic factors in meibomian gland of diabetic mice.Methods Fifty 8-week-old male C57BL/6 mice of clean degree were divided into normal control group(n=20)and diabetes model group(n=30)according to a random table.Diabetes model was established by the intraperitoneal injection of streptozotocin(60 mg/kg,10 mg/ml).Mouse tail vein blood glucose≥16.7 mmol/L was considered as successful modeling.Blood glucose was measured weekly,and body weight was compared between the two groups.Ten mice were randomly selected for fluorescein sodium staining of the cornea to evaluate the integrity of the corneal epithelium from both groups at an interval of 4 weeks.Five mice were randomly selected from the two groups and were sacrificed via anesthesia to collect meibomian gland tissue for hematoxylin and eosin staining in order to observe morphological changes at 8 and 16 weeks after modeling,respectively.At 16 weeks following modeling,mebomian gland of 5 mice randomly selected from both groups was stained with oil red O staining to observe the distribution of lipid.Real-time fluorescence quantitative-PCR was performed to detect the relative expressions of tumor necrosis factor(TNF)-α,pigment epithelium derived factor(PEDF),peroxisome proliferators-activated receptorγ(PPARγ),and adipose differentiation-related protein(ADFP)mRNA in meibomian gland.The use and care of animals complied with the ARVO statement.This study protocol was approved by the Institutional Animal Care and Use Committee of Tianjin Medical University Eye Hospital(No.TJYY20190630009).Results The successful modeling rate of diabetes in mice was 100%,and the survival rate was 83.3%(25/30).The weight was significantly lower and the blood glucose level was higher in diabetes model group at 8 and 16 weeks after modeling in comparison with normal control group(all at P<0.05).There were significant differences in corneal fluorescein staining score among different time points in diabetes model group(F=27.155,P<0.05).In diabetes model group,thinner wall of meibomian gland duct,enlarged lumen of the duct,dilated acini and oil red-stained lipid deposition in most acini were observed.At 16 weeks after modeling,the expressions of TNF-α,and PPARγmRNA in meibomian gland of diabetes model group were 3.33±0.91 and 1.55±0.25,which were significantly higher than 1.00±0.16 and 1.00±0.27 of normal control group(both at P<0.05).The expression of PEDF mRNA in diabetes model group was 0.42±0.08,which was significantly lower than 1.00±0.34 in normal control group(P<0.05).There was no significant difference in the ADFP mRNA expression between the two groups(t=0.943,P=0.38).Conclusions Inflammatory factors and lipid metabolic factors such as TNF-α,PEDF,and PPARγmay be involved in the pathogenesis of meibomian gland dysfunction induced by diabetes.