Effects Of High-concentration Glucose And High-concentration Insulin On Calcium Channel And [Ca²⁺]i In Cultured Müller Cells

In modern china, many people suffered from diabetic, and some patients develop a problem called diabetic retinopathy. Diabetic retinopathy (DR) has become one of four major illnesses which could decrease vision and blindness. Till now, using insulin is still the most credible method to cure diabetic. For the retina cell, a special kind of neuroglia cell, throughout the layer of retina, these cells would be easily affected when the ailment has been taken place in these patients. In the diabetic-retina-model, the increase of Müller cell has been proved. Meantime, proper control with the calcium channel could slower the process and alleviate patients'suffer. Being the conditioner and signal transfer, the concentration of Ca2+ intra-cellule can provide more information than we had expected in the diabetic. Slowly, but with persistent courage, by inspect the concentration of Ca2+ and electrical current, the connection between retina cell with glucose and insulin could deduced. This novel idea would be furthered DR therapy and more experiment investigations on DR are necessary.Objective: Finding the connection between glucose and insulin with retina Müller cell by inspect the concentration of Ca2+ and electrical current.Method:Establish primary culture system of vitro rabbit retina Müller cell by using tissue piece suspension culture, and devide them into normal control, high-concentration glucose group, high-concentration insulin group, high-glucose and high-insulin group.Observe the variation of Ca2+ in retinal Müller cell by the methad of whole cell patch clamp technique, and show the relationship between the variation and influential factors. Detect the change of [Ca2+]i in Müller cell under different cultivante condition by using fluorescent probe technique combined with flow cytometry, and observe ultrastructural change in Müller cell by transmission electron microscope.Result: 1. It is showed that the purity of retinal Müller cell coefficient reach above 95% by contrast phase microscope, HE stain, immunochemical stain, immunofluorescein stain, and transmission electron microscope. 2. The outcomes of whole cell patch clamp technique manifest that, high-concentration glucose group's calcium channel open probability ratio is higher than normal control's. In the third day, the ICa has statistical difference (P<0.05),and in the fifth day, the ICa has obvious statistical difference (P<0.01); In high-concentration insulin group, high-glucose and high-insulin group compaired with normal control, the ICa has no obvious statistical difference (P>0.05); In the third and the fifth day, calcium channel open probability ratio of high-glucose and high-insulin group is lower than that in high-concentration glucose group,and there is an significant difference (P<0.01). These changes have time dependence. 3. The outcomes of fluorescent probe combined with flow cytometer show that:compared with normal control, [Ca2+]i in high-concentration glucose group, high-glucose and high-insulin group both increase; compared with high-concentration glucose group, [Ca2+]i in high-glucose and high-insulin group cut down. 4. The results of transmission electron microscope show that:the microvilli could be seen on the surface of normal control retinal Müller cell. Cytoplasm was abundance, including big nucleus, glycogen granule, rough endoplasmic reticulum, mitochondria, and characteristic intermediate filament(8-10nm). Under high concentration glucose, the numbers of deep staining cells increased, microvilli decreased and became blunt on cell surface. Nucleus shrunk and became anomalous. Euchromatin increased in nucleus. Cytoplasm was dyed deeply, including dilated rough endoplasmic reticulum, swollen mitochondria with broken crest, increased lipid droplet and residual body. Vacuolus could be seen in the cytoplasm. Under high insulin, microvilli could be seen on cell surface. There were residual body, vaculus, swollen mitochondria in cytoplasm. And the rough endoplasmic reticulum was not dilated. Under high glucose and insulin, microvilli decreased on cell surface. Nucleus became a little anomalous. Cytoplasm included residual body, swollen mitochondria, light dilated rough endoplasmic reticulum. Compared with third day, cell culrured in fifth day had more vaculus in cytoplasm.Conclusion: 1. There is L-calcium channels on retina Müller cell membrane. 2. Under high glucose, calcium channels open probability ratio increase conspicuously on Müller cell membrane, and rough endoplasmic reticulum dilate conspicuously in cytoplasm, so [Ca2+]i increase. 3. Under high insulin, calcium channels open probability ratio, [Ca2+]i and rough endoplasmic reticulum does not change noticeable. 4. insulin can depress the negative effect of high glucose on muller cells. 5. muller cells function can be depressed after exposure to high glucose too long time, by changes of ultrastructure.