CAJ | 학술논문

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. 目的觀察色素上皮衍生因子(PEDF)對糖尿病大鼠視網膜Muller細胞穀氨酰胺閤成酶(GS)錶達的影響.方法將Sprague-Dawley大鼠分為模型組、模型對照組、PEDF榦預組(榦預組)、榦預對照組,每組均為8隻大鼠.模型組、榦預組、榦預對照組大鼠鏈脲佐菌素誘導糖尿病大鼠模型.模型組大鼠不作任何榦預,模型對照組為相同月齡的正常大鼠,榦預組大鼠左眼玻璃體腔註射0.1 μg/μl的PEDF10 μl,榦預對照組大鼠左眼玻璃體腔註射相同容積的燐痠鹽緩遲液.採用免疫組織化學法和實時熒光聚閤酶鏈反應(PCR)法檢測視網膜GS和白細胞介素-1β(IL-1β)的錶達變化.將視網膜Muller細胞置于高糖環境下培養,實驗榦預組中加入100 ng/ml PEDF,空白對照組加入相同容積的培養液,24 h後通過蛋白質免疫印跡(Western blot)法和實時熒光PCR法檢測PEDF對Mailer細胞GS和IL-1β錶達的改變.流式細胞儀錨定蛋白-異硫氰痠熒光素和碘化丙啶(Annexin V-FITC-PI)雙染色法檢測100ng/mlPEDF對高糖狀態下Muller細胞凋亡的影響.結果實時熒光PCR法從基因水平和免疫組織化學法從蛋白質水平檢測均顯示,相對于模型對照組大鼠,模型組大鼠視網膜GS錶達降低,而IL-1β的錶達升高,實時熒光PCR法:GS;t=4.23,P<0.01;IL-1β:t=16.73,P<0.01;免疫組織化學法:GS:t=5.13,P<0.01;IL-1β:t=9.32,P<0.01;榦預組大鼠玻璃體腔註射PEDF 48 h後,IL-1β的錶達下降,GS的錶達升高,與榦預對照組比較,實時熒光PCR法:GS:t=3.87,P<0.01;IL-1β:t=3.61,P<0.05;免疫組織化學法:GS:t=3.32,P<0.05;IL-1β:t=2.63,P<0.05.在高糖環境下,通過實時熒光PCR法和Western bot法檢測均顯示PEDF可以下調IL-1β的錶達,而上調GS的錶達,與空白對照組比較,實時熒光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.流式細胞儀檢測結果顯示,PEDF可以抑製高糖環境下Muller細胞的凋亡,實驗組凋亡率與空白對照組凋亡率比較,差異有統計學意義(t=3.21,P<0.05).結論對于糖尿病大鼠,PEDF可能通過下調視網膜Muller細胞中IL-1β的錶達來上調GS的錶達,從而改善穀氨痠循環,抑製神經節細胞的死亡.
목적 관찰색소상피연생인자(PEDF)대당뇨병대서시망막Muller세포곡안선알합성매(GS)표체적영향.방법 장Sprague-Dawley대서분위모형조、모형대조조、PEDF간예조(간예조)、간예대조조,매조균위8지대서.모형조、간예조、간예대조조대서련뇨좌균소유도당뇨병대서모형.모형조대서불작임하간예,모형대조조위상동월령적정상대서,간예조대서좌안파리체강주사0.1 μg/μl적PEDF10 μl,간예대조조대서좌안파리체강주사상동용적적린산염완충액.채용면역조직화학법화실시형광취합매련반응(PCR)법검측시망막GS화백세포개소-1β(IL-1β)적표체변화.장시망막Muller세포치우고당배경하배양,실험간예조중가입100 ng/ml PEDF,공백대조조가입상동용적적배양액,24 h후통과단백질면역인적(Western blot)법화실시형광PCR법검측PEDF대Mailer세포GS화IL-1β표체적개변.류식세포의묘정단백-이류청산형광소화전화병정(Annexin V-FITC-PI)쌍염색법검측100ng/mlPEDF대고당상태하Muller세포조망적영향.결과 실시형광PCR법종기인수평화면역조직화학법종단백질수평검측균현시,상대우모형대조조대서,모형조대서시망막GS표체강저,이IL-1β적표체승고,실시형광PCR법:GS;t=4.23,P<0.01;IL-1β:t=16.73,P<0.01;면역조직화학법:GS:t=5.13,P<0.01;IL-1β:t=9.32,P<0.01;간예조대서파리체강주사PEDF 48 h후,IL-1β적표체하강,GS적표체승고,여간예대조조비교,실시형광PCR법:GS:t=3.87,P<0.01;IL-1β:t=3.61,P<0.05;면역조직화학법:GS:t=3.32,P<0.05;IL-1β:t=2.63,P<0.05.재고당배경하,통과실시형광PCR법화Western bot법검측균현시PEDF가이하조IL-1β적표체,이상조GS적표체,여공백대조조비교,실시형광PCR법: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.류식세포의검측결과현시,PEDF가이억제고당배경하Muller세포적조망,실험조조망솔여공백대조조조망솔비교,차이유통계학의의(t=3.21,P<0.05).결론 대우당뇨병대서,PEDF가능통과하조시망막Muller세포중IL-1β적표체래상조GS적표체,종이개선곡안산순배,억제신경절세포적사망.
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.