Intervention Effect Of Calpain Inhabitor On Nscs Injury Induced By Acrylamide

Acrylamide (acrylamide,ACR) is a kind of nerve poison that has confirmed by relevant international organizations.The poisoning patients are mainly the operators producting or using the ACR monomer. Toxication particularly easily appears in the conditions of poor ventilation or lack of personal protection. In the occupational exposure persons,such as acrylamide production, polyacrylamide synthesis, sewage treatment, oil drilling and grouting construction, the occupational harm are mainly skin lesions and Nervous System damage.The Nervous System damage includes the Central Nervous System (CNS) and the Peripheral Nervous System (PNS) damage.90%of ACR monomer are used in the production of polyacrylamide (Polyaerylamide, PACR), in recent years, the oil industry has developed rapidly,the demand of PACR increases unceasingly.In recent years, the oil industry has developed rapidly,then the demand of ACR increases, thus the harm of ACR is biger and biger.The study of ACR is more than before, but the cellular and molecular mechanisms of ACR have not been fully elucidated.Some scholars think that ACR poisoning is caused by the axoplasmic transport damage.Cytoskeleton plays an important role in the axoplasmic transport, and ACR comes into play by acting on the axon cytoskeleton protein in different composition and then influences axoplasmic transport, resulting function change of the distal axon and nerve damage. The aggregation or degradation of cytoskeleton protein could be caused by dependent protease (Calpain) decomposes cytoskeleton protein.Calpain plays an important role by influencing the skeleton protein in the pathophysiological process of the nervous system. At present, the rise of intracellular calcium ion levels after CNS injury can enhance the activity of Calpain, and then selectively degrade myelin protein and various cytoskeletal protein. Some scholars by means of immunohistochemical technology found that the expression of Calpain enhanced after spinal cord injury.Experiment selected neural stem cells (neural stem cells, NSCs) in vitro studies. NSCs had multi-directional differentiation potential, in the study of nervous system toxicity representative; NSCs were able to self proliferation, could provide a large number of cells the experiment required.Take spinal cord Dorsal Root ganglia (Dorsal Root Ganglion, DRG) from Wistar rat born within24h to obtain the original generation cells and appraised them. Obtain stable cell lines. Then infecte cells with ACR,includes the control group, the infected group(ACR) and intervention group(ACR+MDL-28170).Measure absorbance values with the thiazole blue colorimetric method (determined by MTT).Calculate the cell survival rate to establish the infected model of NSCs injury induced by ACR.On the basis of this model intervene with calcium protease inhibitors MDL-28170, then intervention model was established.By testing intracellular calcium ion concentration and Calpain activity to further determine the intervention effect of calpain inhibitor on NSCs injury induced by ACR.Objective1. To build the model of ACR infected the neural stem cell.On the basis of this model build the model that discusses the intervention effect of calpain inhibitor.2. By measuring the concentration of the intracellular calcium and the activity of Calpain to verify the success of the model, to confirm the intervention effect of calpain inhibitor on NSCs induced by acrylamide. To provide the reference for subsequent research on mechanism of NF change and prevention of ACR exposed workers’NSCs injury.Methods1. Strip the spinal cord dorsal root ganglion of rat born within24h under aseptic conditions, inoculated after trypsin digestion. Detect three indicators including nests protein (nestin), specificity enolization enzyme of neurons (NSE)and glial fibers acidic protein (GFAP) by immunohistochemical method to identify neural stem cells. Obtain the stable cell line by cell subculture.2. Medicate with ACR to the cell line that had established. Then determine the appropriate dose and time according to the cell survival rate tested by MTT. A cell model was established through the toxicity test. On the basis of this model, establish a intervention model in a similar way.3. Use the Fura-2/AM probe and calpain substrate11to detect the concentration of the intracellular calcium and the activity of Calpain.Results1. The capture and identification of primary cellGather the spinal dorsal root ganglion to culture neural stem cells and then identificate them Immunocytochemistry was performed to identify the cells. Nestin was identificated by antibody of nestin and the result was positive.The same source of cells could be induced to differentiate into NSE positive neurons. GFAP was positive too.2. The establishment of infect and intervention model(1) Determine the ACR initial infected concentration were0,1400,2800,4200,5600μmol/L. The group with dose of2800μmol/L had difference with the control group(P<0.05) after24hours and the group with1400μmol/L had difference with the control group(P<0.05) after48hours. Adjust the dose according to the result,24-hour infected dose were adjusted for0,2240,2520,2800,3080μmol/L.The group with dose of224μmol/L had difference with the control groups<0.05).Infected48hours dose were adjusted for0,700,840,980,1120,1400μmol/L. The group with dose of700μmol/L had difference with the control group(.P<0.05). According to the result to further adjust the dose,24hour infected dose were0,1050,1400,1750,2100μmol/L. The group with dose of2100μmol/L had difference with the control group(P <0.05). Infected dose adjustment for48hours were0,140,350,560,700μmol/L. The group with dose of700μmol/L had difference with the control group(P<0.05).2100μmol/L reacted for24hours and700μmol/L reacted for48hours could be classified as the experimental infected dose and time.(2) Choose that the ACR infected in2100umol/L for24hours and700μmol/L for48hours. At the same time,the calpain inhibitors intervented with0,65,130,195,260μmol/L concentration.Intervention results of24hours:Compared with the control group,the absorbance value of the else groups were all lower than the control group, the difference was significant (P<0.05).Compared with the infected group, the absorbance value of65,130μmol/L intervention group were higher, the difference was significant (P<0.05); the absorbance value of195,260umol/L intervention group were lower, the difference was significant (P<0.05).Intervention results of48hours-.Compared with the control group,the absorbance value of the else groups were all lower than the control group, the difference was significant (P<0.05).Compared with the infected group, the absorbance value of65,130μmol/L intervention group were higher, the difference was significant (P<0.05);there was not difference between the195μmol/L group and infected group(P>0.05); the absorbance value of260μmol/L intervention group was lower, the difference was significant (P<0.05).3. The result of intracellular calcium ion concentrationCompared with control group, intracellular calcium ion concentration of infected group was higher, the difference was significant (P<0.05); there was not difference between intervention group and control group(P>0.05).Compared with the infected group, intracellular calcium ion concentration of intervention group was lower than the infected group, the difference was significant (P<0.05).4. The result of the Calpain activityCompared with control group, the Calpain activity of infected group was higher, the difference was significant (P<0.05); the Calpain activity of intervention group was higher than the control group, the difference was statistically significant (P<0.05).Compared with the infected group, the Calpain activity of intervention group was lower than the infected group, the difference was significant (P<0.05).Conclusion1. The NSCs were successfully authenticated. The model of ACR acting on neural stem cells was established. And the intervention model of calpain inhibitor was established.2. The damage of ACR caused the decrease of cell survival rate, the rise of intracellular calcium ions levels,the enhancement of Calpain activity.MDL-28170could inhibit this injury, effectively increase the cell survival rate, reduce the levels of intracellular calcium ions and Calpain activity.3. Confirm that the calpain inhibitor had intervention effect on NSCs injury induced by acrylamide.