白细胞介素-6对糖尿病小鼠角膜缘干细胞活化的促进作用和角膜上皮愈合的加速作用

背景 白细胞介素(IL)-6既介导炎症反应过程又在损伤修复中发挥重要作用,但其具体机制及其在糖尿病角膜组织损伤修复中的作用值得探讨. 目的 探讨IL-6在正常和糖尿病小鼠角膜缘干细胞活化和角膜上皮修复过程中的作用及其机制.方法 正常6～8周龄C57B L/6小鼠52只,采用随机数字表法随机分为正常对照鼠32只和糖尿病模型鼠20只,采用50 mg/kg链脲佐菌素连续腹腔内注射5d的方法诱导建立小鼠糖尿病模型.正常对照小鼠和糖尿病模型小鼠均行角膜上皮刮除术,然后分别于刮除后即刻和48 h结膜下注射IL-6或等容量PBS,采用荧光素染色法评价随时间延长的角膜上皮的愈合情况.体外培养小鼠角膜上皮干/祖细胞(TKE2细胞系),采用结晶紫染色法评估不同质量浓度IL-6处理后细胞克隆率(CFE),并与空白对照组进行比较;采用免疫荧光检测法和Western blot法检测细胞和小鼠再生上皮中干细胞标志物△NP63、Ki67的表达以及关键转录因子STAT3磷酸化水平;采用实时荧光定量PCR法和ELISA法检测小鼠角膜再生上皮中IL-6的mRNA及蛋白水平.结果 角膜荧光素染色检查显示,正常对照小鼠和糖尿病模型小鼠PBS注射组与IL-6注射组注射后24、48和72 h残留角膜上皮缺损面积占原始缺损面积的百分比随角膜上皮损伤后时间延长均明显缩小,同时间点IL-6注射组角膜上皮缺损面积均明显小于PBS注射组,组间总体差异均有统计学意义(正常对照组:F分组=19.982,P＜0.01;F时间=589.350,P＜0.01;糖尿病组:F分组=25.411,P＜0.01;F时间=334.807,P＜0.01).空白对照组及10、20、50、100 ng/ml IL-6处理组CFE分别为(13.23±1.12)％、(15.87±1.30)％、(21.69±1.62)％、(25.33±1.28)％和(18.67±1.54)％,随着IL-6质量浓度的增加CFE逐渐增加,总体比较差异有统计学意义(F=35.547,P＜0.01).50 ng/ml IL-6处理5、10、15、30和60 min细胞中△NP63、Ki67和p-STAT3蛋白的相对表达量随着处理时间的延长均逐渐增加,各时间点细胞中△NP63、Ki67和p-STAT3蛋白的相对表达量均明显高于空白对照组,差异均有统计学意义(均P＜0.05).糖尿病组和正常对照组小鼠角膜上皮刮除后24 h的再生上皮中IL-6 mRNA相对表达量分别为0.45±0.21和1.00±0.16,糖尿病组小鼠角膜再生上皮中IL-6 mRNA相对表达量较正常对照组小鼠下降56％,差异有统计学意义(t=3.42,P=0.03),糖尿病小鼠角膜上皮刮除后48 h的再生上皮中IL-6蛋白质量浓度为(257±12)ng/μl,明显低于正常对照组小鼠的(323±17) ng/μl,差异有统计学意义(t=5.60,P＜0.01). 结论 IL-6能够通过激活STAT3信号通路促进正常和糖尿病小鼠角膜缘干细胞的活化和增生,进而促进角膜上皮修复,而封闭内源性IL-6则会延迟小鼠角膜上皮的修复.

Effects of interleukin-6 in promoting corneal epithelial stem/progenitor cell regeneration and accelerating corneal epithelial wound healing in diabetic mouse

Background Interleukin-6 (IL-6) is a pleiotropic cytokine involving in inflammation and wound healing.Previous report found that IL-6 increases phosphorylated STAT3 (p-STAT3) level and promotes corneal epithelial wound healing by stimulating migration.However,the essential role of IL-6 in corneal epithelial wound healing and the expression changes in diabetic mellitus remains unknown.Objective This study was to explore the roles of IL-6 in corneal epithelial proliferation and wound healing in both normal and diabetic mice.Methods Fifty-two normal C57BL/6 mice were randomized into normal control group (32 mice) and diabetic group (20 mice).Type 1 diabetic mellitus was induced by intraperitoneal injections of 50 mg/kg streptozotocin once per day for consecutive 5 days in the mice of the diabetic group.Whole corneal epithelium was scraped in all mice,and the corneal epithelial defect area was examined by fluorescein staining in 24,48 and 72 hours after corneal epithelium removal.Recombinant mouse IL-6 or anti-IL-6 blocking antibody of 5 μl were subconjunctivally injected according to the grouping and contrasted with PBS injection group or isotype control antibody group,respectively.TKE2 cells,a mouse corneal epithelial stem/progenitor cell line,were trypsinized and incubated in the KSFM with different concentrations of IL-6 or without IL-6,and colony formation efficency (CFE) was examined by crystal violet staining.The expressions of △NP63 and Ki67,specific makers of stem cells,were detected by immunofluorescine technology.The expressions of △NP63,Ki67 and p-STAT3 proteins were assayed in the cells by Western blot,respectively.The expression of IL-6 mRNA and protein in the regenerated corneal epithelium was detected by real time quantitative PCR and ELISA.The use and care of the mice complied with the Statement of Association for Research in Vision and Ophthalmology.Results The percentage of residual corneal epithelium defect area with initial detect area was gradually shrinked over time after PBS and IL-6 injection in both normal control mice and diabetic mice,and the percentage of residual corneal epithelium defect area was significantly reduced in the IL-6 injected group compared with the PBS injected group (normal control group:Fgroup =19.982,P ＜ 0.01;Ftime =589.350,P ＜ 0.01;Diabetic group:Fgroup =25.411,P＜0.01;Ftime =334.807,P＜0.01).The CFE was (13.23± 1.12)％,(15.87± 1.30)％,(21.69±1.62)％,(25.33±1.28)％ and (18.67±1.54)％ in the blank control group and 10,20,50,100 ng/ml IL-6-treated groups,respectively,showing a gradual increase of CFE dependent upon IL-6 concetrations (F =35.547,P＜0.01).The expressions of △NP63,Ki67,p-STAT3 proteins in the cells were gradually increased over time after 50 ng/ml IL-6 treated for 5,10,15,30 and 60 minutes,and the relative expression level of the cytokines was significnatly higher in the IL-6 cultured groups than that without IL-6 culture group (all at P＜0.05).The relative expression of IL-6 mRNA in the regenerated corneal epithelilum was 0.45±0.21 and 1.00±0.16 in the diabetic group and normal control group,respectively,and compared with the normal control group,the expression of IL-6 mRNA reduced by 56％ (t=3.42,P=0.03).The content of IL-6 protein in regenerated corneal epithelium of the diabetic group was (257±12) ng/μl,which was significantly lower than (323 ± 17) ng/μl of the normal control group (t =5.60,P＜0.01).Conclusions IL-6 promotes the proliferation and regeneration of corneal limbal stem cells to repair defected corneal epithelium by activating STAT3 signaling pathway in both normal and diabetic mice,while the blocking of endogenous IL-6 impairs the corneal epithelial cell activation and wound healing.