Effects And Mechanism Of Fluctuate Glucose On Rabbit Retinal Müller Cells

Objective: Rabbit retinal Müller cells were cultured in vitro，Simulated thesituation of diabetic’s blood glucose sharp fluctuation, observing glucosestability on the vitality and inducible nitric oxide synthase expression in rabbitretinal Müller cells. To explore the function and mechanism of fluctuate glucosein diabetic retinopathy. Providing experiment basis for the clinical treatment ofdiabetes and its complications.Method: Using enzymatic digestion method cultured rabbit retinal Müllercells. Cultured cells were identified and assessed purity by electron microscopeand immunofluorescence techniques. The good state P2cells were used inexperiment. The cells were divided into five groups: normal control group(5.5mmol/L glucose), constant high glucose group (30mmol/L glucose),fluctuate glucose group (30mmol/l glucose for three hours, then cultured in5.5mmol/L glucose for two hours, such alternation was repeated three times aday and the cells were cultured in30mmol/L glucose in the night), osmolalitycontrol group one(5.5mmol/Lglucose+24.5mmol/L mannitol), osmolalitycontrol group two ((5.5mmol/Lglucose+24.5mmol/Lmannitol) for threehours, then cultured in5.5mmol/L glucose for two hours, such alternation wasrepeated three times a day and the cells were cultured in(5.5mmol/Lglucose+24.5mmol/L mannitol) in the night). Three days later, MTT assay was used toobserve the vitality of Müller cells. Immunofluorescence was used to detectcells’ inducible nitric oxide synthase (iNOS), measure its average fluorescencedensity by the image analysis software.Results:(一) After using enzymatic digestion method culture rabbit retinalMüller cells, some adherent cells can be seen in the bottom after2-3days. Cellswere transparent, rounded or slightly present fusiform, some cells can be seemsome processes. About16days, Cells covered the entire bottom of the bottle, theshape was relatively uniform, irregular in shape, showing cell processes.Electron microscope showed Cytoplasm is rich in8-10nm microfilaments andvarious organelles; cellular retinaldehyde binding protein and glutaminesynthetase were green and red fluorescent in Müller cells by the method ofimmunofluorescence. The positive rates were more than95%.(二) Comparingwith normal control group and two osmolality control groups, the cells’vitality in fluctuate glucose groups (5.5/30mmol/L) and constant highglucose groups (30mmol/L) were declined after three days (p<0.01), andthe iNOS expression were increased(p<0.01). These changes were moreobvious in fluctuate glucose groups. Normal control group and twoosmolality control groups’ cell vitality and iNOS expression have nosignificance(p﹥0.05). Conclusions:(一) Using enzymatic digestion method culture rabbit retinalMüller cells can shorten incubation time, obtain a large number of high-puritycell. It can better meet the needs of the experimental process;(二) Highconcentration of glucose possibly by enhancing the expression of iNOS todamage the function of Müller cells, this damage is related to sugarconcentration, instead of osmoti pressure. And fluctuate glucose is morecytotoxicity than constant high glucose, it suggested that fluctuate glucoseplay an important role in the occurrence and development of diabeticretinopathy.