A Preliminary Study On The Expression Of Metrnl In Schwann Cells And Its Biological Effects

Objectives:1.To identify the expression of Metrnl in rat sciatic nerve and primary Schwann cells from non-diabetic normal rats and diabetic neuropathic rats.respectively.2.To observe the proliferation of Schwann cells derived from sciatic nerve in different groups.23.To observe the expression of Metrnl and NGF in Schwann cells derived from sciatic nerve from different groups.4.To observe the effect on the proliferation of Schwann cells after endogenous Metrnl expression is depressed by siRNA5.To observe the effect on the expression of endogenous NGF after endogenous Metrnl expression is depressed by siRNAMethods:1.12 male Sprague-Dawley rats were randomly divided into the normal group(n?6)and the diabetic group(n?6).The diabetic rats were induced by intraperitoneal injection of STZ and verified by the blood glucose(over 16.7mmol/1)after 72 hours.During the experiment,blood glucose and body weight of all rats were recorded weekly.2.The sciatic nerve was stimulated proximally at the sciatic notch and distally at the ankle via bipolar electrodes with supramaximal stimuli(8 V)at 20 Hz.The latencies of the compound muscle action potentials were recorded via bipolar electrodes from the first interosseous muscle of the hindpaw and measured from the stimulus artifact to the onset of the negative M-wave deflection.MNCV was calculated by subtracting the distal latency from the proximal latency,and the result was divided into the distance between the stimulating and recording electrode.3.Under aseptic conditions,sciatic nerves were obtained from Sprague-Dawley rats.The epineurium was stripped off using fine-pointed forceps,and the sciatic nerve was cut into 2-3 mm fragments.Then nerve fragments were digested in 0.25%trypsin and 0.05%collagenase ? solution in the incubator for 90 min.The cell suspension was then washed using stop medium and centrifuged at 1200rpm for 6 min.The supernatant was discarded and the deposit was re-suspended with Schwann cell-culture medium.Cells were then cultured in plates pre-coated with laminin and poly-lysine.Cells were incubated with cytarabine in the first 3 days and replaced with complete medium after 3 days.When the cells began to growth stably,the purity of Schwann cells were identified by cellular immunofluorescence staining.4.The expression of Metnl in sciatic nerve of Sprague-Dawley rats was identified by inmunohistochemical staining.The expression of Metml in primary Schwann cells was identified by immunofluorescence staining.5.Schwann cells were divided into 3 groups using complete medium:high-glucose group(glucose concentration 45mmol/l,Schwann cells from sciatic nerves of normal rats),normal-glucose group(glucose concentration 5.56mmol/l,Schwann cells from sciatic nerves of diabetic rats),hypertonic mannitol group(5.56mmol/l glucose+39.5mmol/l mannitol,Schwann cells from sciatic nerves of normal rats).When the confluency of cells reached 80%,the medium was changed to high-glucose DMEM(glucose concentration 45mmol/1),normal-glucose DMEM(glucose concentration 5.6mmol/1)and hypertonic mannitol DMEM(5.56mmol/1 glucose+39.5mmol/1 mannitol),separately.After 48 hours,Schwann cells and medium were collected and the expression of Metrnl as well as NGF measured by Real-Time PCR,Western blot and ELISA.6.Schwann cells,when reaching 60-70%confluency,were transfected with Metrnl siRNA in Lipofectamine(?)3000,according to manufacturer's instructions.One sequence with better interfering effect on the expression of Metml was evaluated through Real-Time PCR and Western blot.7.The Schwann cells transfected with siRNA were divided into two groups:siRNA-M-2 groups and comtrol groups.Schwann cells from different groups of sciatic nerve were divided into three groups according to the above methods:high-glucose group from diabetic neuropathic rats,normal-glucose group from normal rats and hyperosmotic mannitol group,the OD value at 450 nm of different cells at different time was measured using CCK8 method.8.The siRNA-transfected Schwann cells were divided into two groups:siRNA-M-2 group and NC group.The supernatant was discarded and replaced with DMEM(glucose concentration 5.56mmol/1)for 24 hours.The cells were then collected and the apoptosis of cells were measured by flow cytometry.Results:1.The expression of Metml was identified in rat sciatic nerve and primary Schwann cells3.Compared with the normal-glucose group,the expression of Metml and NGF in the Schwann cells of high-glucose group from diabetic neuropathic rats was decreased,while the expression of Metml and NGF in the Schwann cells of normal-glucose group from normal rats and the hypertonic mannitol group showed no statistical significance.4.Compared with that of the normal-glucose group cells,the proliferation of Schwann cells in the high-glucose group from diabetic neuropathic rats was decreased,while the proliferation of Schwann cells showed no statistical significance between the normal-glucose group and the hypertonic mannitol group from normal rats.5.Compared with that of Schwann cells in NC group,the proliferation of Schwann cells in siRNA-M-2 group was decreased,but the apoptotic rate was not statistically different between cells in these two groups.Conclusion:1.Metml is expressed in rat Schwann cells and the expression is decreased in diabetes.2.Silence of Metml in Schwann cells inhibites the proliferation of Schwann cells,indicating that depressed-Metml is related to reduced-proliferation od Schwann cells,at least in part contributing to dysfunctions of sciatic nerves in diabetic rats.3.Silence of Metmrn in Schwann cells does not affect their survivability,which is probably due to compensatory up-regulation of NGF.