色素上皮衍生因子对糖尿病大鼠视网膜谷氨酰胺合成酶表达的影响

The effect of pigment epithelium-derived factor on expression of glutamine synthetase in diabetic rat retina

目的 观察色素上皮衍生因子（PEDF）对糖尿病大鼠视网膜Muller细胞谷氨酰胺合成酶（GS）表达的影响.方法 将Sprague-Dawley大鼠分为模型组、模型对照组、PEDF干预组（干预组）、干预对照组,每组均为8只大鼠.模型组、干预组、干预对照组大鼠链脲佐菌素诱导糖尿病大鼠模型.模型组大鼠不作任何干预,模型对照组为相同月龄的正常大鼠,干预组大鼠左眼玻璃体腔注射0.1 μg/μl的PEDF10 μl,干预对照组大鼠左眼玻璃体腔注射相同容积的磷酸盐缓冲液.采用免疫组织化学法和实时荧光聚合酶链反应（PCR）法检测视网膜GS和白细胞介素-1β（IL-1β）的表达变化.将视网膜Muller细胞置于高糖环境下培养,实验干预组中加入100 ng/ml PEDF,空白对照组加入相同容积的培养液,24 h后通过蛋白质免疫印迹（Western blot）法和实时荧光PCR法检测PEDF对Mailer细胞GS和IL-1β表达的改变.流式细胞仪锚定蛋白-异硫氰酸荧光素和碘化丙啶（Annexin V-FITC-PI）双染色法检测100ng/mlPEDF对高糖状态下Muller细胞凋亡的影响.结果 实时荧光PCR法从基因水平和免疫组织化学法从蛋白质水平检测均显示,相对于模型对照组大鼠,模型组大鼠视网膜GS表达降低,而IL-1β的表达升高,实时荧光PCR法：GS t=4.23,P〈0.01 IL-1β：t=16.73,P〈0.01 免疫组织化学法：GS：t=5.13,P〈0.01 IL-1β：t=9.32,P〈0.01 干预组大鼠玻璃体腔注射PEDF 48 h后,IL-1β的表达下降,GS的表达升高,与干预对照组比较,实时荧光PCR法：GS：t=3.87,P〈0.01 IL-1β：t=3.61,P〈0.05 免疫组织化学法：GS：t=3.32,P〈0.05 IL-1β：t=2.63,P〈0.05.在高糖环境下,通过实时荧光PCR法和Western bot法检测均显示PEDF可以下调IL-1β的表达,而上调GS的表达,与空白对照组比较,实时荧光PCR法：GS：t=2.89,P〈0.05 IL-1β：t=3.37,P〈0.05 Western blot：GS：t=2.66,P〈0.05 IL-1β：t=3.23,P〈0.05.流式细胞仪检测结果显示,PEDF可以抑制高糖环境下Muller细胞的凋亡,实验组凋亡率与空白对照组凋亡率比较,差异有统计学意义（t=3.21,P〈0.05）.结论 对于糖尿病大鼠,PEDF可能通过下调视网膜Muller细胞中IL-1β的表达来上调GS的表达,从而改善谷氨酸循环,抑制神经节细胞的死亡.

Objective To investigate the effect of pigment epithelium-derived factor （PEDF）on the expression of glutamine synthetase in retinal Mailer cells of diabetic rats. Methods Diabetic rats were induced with streptozotocin injection. Before and after injection of 10 μ1 （0. 1 μg/μl） PEDF （experimental group） or 10 μl PBS （control group） into the vitreous cavities of diabetic rats respectively for 48 hours, the expressions of GS and IL-1β in retina were analyzed by immunohistochemistry and real time RT-PCR techniques. After being treated with 100 ng/ml PEDF for 24 hours in high glucose conditions, the expressions of GS and IL-1β in cultured MOiler ceils were studied by western blot and real time RT-PCR techniques. Apoptosis was analyzed by flow cytometry after Annexin V-fluorescein isothiocyanate/Propidium idoium （Annexin V-FITC/PI） staining. Results By immunohistochemistry （the protein level） and real time RT-PCR （the mRNA level）, it was found that the expression of GS decreased and the expression of IL-1β increased obviously （real time RT-PCR：GS： t=4. 23, P〈0. 01 IL-1β： t= 16. 73,P〈0. 01 immunohistochemistry： GS： t=5.13,P〈0. 01 IL-1β： t= 9. 32, P〈0. 01） in diabetic rats. After injection of 10 μl （0. 1 μg/μl） PEDF into the vitreous cavities of diabetic rats for 48 hours, it was found that the expression of GS increased and the expression of IL-1β decreased significantly（RT-PCR GS： t= 3. 87, P〈0.01 IL-1β： t= 3. 61,P〈0. 05 immunohistochemistry：GS： t = 3. 32, P〈 0.05 IL-1β： t = 2. 63, P〈0.05）. Under high glucose conditions, 100 ng/ml PEDF induced decreasing expression of IL-1β and increasing expression of GS significantly （RT-PCR：GS： t=2.89, P〈0. 05 IL-1β： t=3. 37, P〈0. 05 Western blot：GS： t=2.66, P〈 0. 05 IL-1β： t=3.23, P〈0. 05）. Apoptosis of Muller cells under high glucose conditions was inhibited significantly by the treatment with 100 nmol/ml PEDF （t=3.21, P〈0. 05）. Conclusions In diabetic rats, PEDF may decrease expression of IL-1β in rat retinal Mailer cells, which may result in increasing expression of GS. To some degree, it inhibits possibly the death of retinal ganglion cells.