钙蛋白酶抑制剂拮抗丙烯酰胺染毒大鼠脊髓神经丝变化及其机制

发布时间：2018-05-29 06:49

本文选题：丙烯酰胺 + 钙蛋白酶抑制剂　；参考：《山东大学》2014年硕士论文

【摘要】：目的丙烯酰胺(acrylamide, ACR)应用广泛,以它为单体作为原料合成的工业产品多达百种。ACR能诱发中枢-周围神经病。临床表现有出汗、手脚感觉异常、腿部痉挛、白指、皮肤刺激和皮肤剥落等,之后表现为骨骼肌无力和四肢麻痹。ACR诱导的病变尚无定论,国内外研究多集中于神经元轴索,但是近年来国内外学者研究表明ACR诱导的细胞Ca2+内流,钙依赖性蛋白酶活性升高,病理特征显示轴突内肿胀和神经丝堆积。本研究之前以建立ACR中毒模型,发现组织内的神经丝(neurofilaments, NFs)发生了明显改变。但其中NFs是如何改变的尚不清楚。我们假设这种NFs的改变与Calpain有关,为验证这种假设,本研究通过建立ACR中毒神经病大鼠模型及钙蛋白酶抑制剂(Calpeptin, CP)干预模型,测定大鼠神经行为学的变化及脊髓组织中Ca2+、Calpain活性、NFs蛋白表达水平,为研究ACR引起的神经病变的发病机制和预防措施提供理论依据。方法 1动物模型及行为观察将30只成年雌性Wistar大鼠适应性饲养一周后,随机分为3组,即对照组、模型组和干预组(n=10)。ACR溶解生理盐水中,模型组和干预组按30mg/kg·bw经腹腔注射染毒,对照组给予等体积生理盐水。干预组在注射ACR1h后,给予Calpeptin200μg/kg-bw,每3次／周,连续4周。于第4周,断头处死大鼠,取大脑、脊髓备用。每周测体重及神经行为学指标1次。 2神经行为学指标测定给抑制剂期间测定不同时间点各组大鼠神经行为学指标：步态评分、后肢撑力指数。 3分离脊髓,胰酶消化后,Fura-2/AM装载后用荧光分光光度计测定钙离子含量。 4取脊髓经组织匀浆上清,加Calpain substrate II,用酶标仪测定荧光强度,得出Calpain活性。 5蛋白免疫印迹(Western blotting)测定脊髓组织中的NF-L、NF-M蛋白表达水平。结果 1大鼠一般状况变化对照组大鼠体重平稳增长；模型组大鼠体重增长较为缓慢,两者体重于第2周开始出现显著差异(P0.05)；干预组大鼠体重在第2周时体重显著高于模型组(P0.05)。 2大鼠神经行为学改变模型组大鼠腹部着地,两腿叉开平铺、划行、呈鸭步,干预组出现不同程度的步态异常,后肢间距增宽,但异常程度较模型组轻,其中模型组步态评分呈3分的占60%,干预组2-3分大鼠分别占80%和20%,与模型组比差异有统计学意义(P0.01)。后肢撑力实验结果显示,模型组大鼠后肢间距比对照组明显增宽(P0.01),干预组大鼠后肢间距明显比模型组降低,到第4周,干预组大鼠后肢间距比模型组降低,差异有统计学意义P0.05)。 3大鼠脊髓组织Ca2+变化与对照组相比,模型组和干预组分别升高了25.34%和17.03%,差异有统计学意义(P0.05)；与模型组比较,干预组降低了6.63%差异有统计学意义(P0.05)。 4大鼠Calpain活性的改变与对照组比,模型组脊髓Calpain活性升高了33.33%(P0.01),干预组升高了15.87%(P0.01)；与模型组比,干预组脊髓Calpain活性降低了13.10%(P0.05)。 5大鼠脊髓神经丝蛋白改变与对照组相比,模型组NF-L、NF-M分别升高了30.0%、60.9%(P0.05),干预组NF-L、NF-M分别升高了14.1%和25.9%(P0.05)。与模型组相比,干预组的NF-L、NF-M分别下降了12.6%和21.7%(P0.05)。结论 1ACR可引起大鼠的神经行为功能异常, CP能拮抗其异常改变。 2ACR引起脊髓中Ca2+浓度、Calpain活性、NFs表达增高,CP抑制了Ca2+浓度、Calpain活性、NFs的升高,提示CP对ACR染毒大鼠具有神经保护作用。 3CP能降低ACR引起的NFs的堆积程度,提示ACR的神经毒性作用机制可能与calpain的变化相关
[Abstract]:objective
Acrylamide (ACR) is widely used as an industrial product synthesized by it as a monomer. Up to 100 kinds of.ACR can induce central peripheral neuropathy. Clinical manifestations include sweating, abnormal hand and foot sensation, leg spasm, white finger, skin irritation and skin exfoliation, followed by.ACR induced lesions of skeletal muscle weakness and extremities. The study at home and abroad is mostly concentrated on the axonal of neuron, but in recent years, domestic and foreign scholars have shown that ACR induced Ca2+ internal flow and calcium dependent protease activity increased. Pathological features show the swelling of the axon and the accumulation of nerve filament. Before this study, the model of ACR poisoning was established and the nerve filaments (neurofilaments, NFs) were found in the tissue. There has been a significant change. But it is not clear how NFs changes. We assume that the changes in NFs are related to Calpain. In order to verify this hypothesis, this study is to determine the neurobehavioral changes in rats and the Ca2+, Cal in spinal cord tissue by establishing ACR poisoned neuropathy rat model and calsin inhibitor (Calpeptin, CP) intervention model. Pain activity and NFs protein expression level provide a theoretical basis for studying the pathogenesis and preventive measures of ACR induced neuropathy.
Method
1 animal model and behavior observation were adapted to 30 adult female Wistar rats for one week. They were randomly divided into 3 groups, namely the control group, the model group and the intervention group (n=10).ACR dissolved saline. The model group and the intervention group were injected with 30mg/kg. BW by intraperitoneal injection, and the control group was given equal volume of saline. The intervention group was given Calp after the injection of Calp. Eptin200 g/kg-bw, 3 times per week, for 4 weeks. After fourth weeks, the rats were killed by decapitation. The brain and spinal cord were collected. Body weight and neurobehavioral indexes were measured 1 times a week.
2 neurobehavioral indexes were measured at different time points during the inhibition period.
3 the spinal cord was separated. After pancreatin digestion, Fura-2/AM was loaded and the content of calcium was determined by spectrofluorometer.
4 spinal cord tissue homogenate supernatant, plus Calpain substrate II, the fluorescence intensity was measured by enzyme labelling instrument, and Calpain activity was obtained.
5 protein immunoblotting (Western blotting) was used to detect the expression level of NF-L and NF-M protein in spinal cord tissue.
Result
The body weight of the 1 rats in the control group increased steadily, and the weight growth of the model group was slower, and the weight of the two groups began to appear significant difference at the beginning of the second week (P0.05), and the weight of the rats in the intervention group was significantly higher than the model group at second weeks (P0.05).
The 2 rats in the neurobehavioral change model group were on the ground in the abdomen, the legs were paved and crossed, and the duck steps were crossed. The intervention group had different degrees of gait abnormality and the width of the hind limbs widened, but the degree of abnormality was lighter than the model group. Among them, the gait score of the model group was 3 and 60%, and the 2-3 rats in the intervention group were 80% and 20% respectively, and there was a statistical difference compared with the model group. Study significance (P0.01). The results of hind limb strength test showed that the distance of hind limbs of rats in model group was significantly wider than that of control group (P0.01), and the distance between hind limbs of rats in intervention group was significantly lower than that of model group. After fourth weeks, the distance of hind limbs of rats in intervention group was lower than that of model group, and the difference was statistically significant P0.05).
The changes of Ca2+ in the spinal cord tissue of the 3 rats were 25.34% and 17.03% in the model group and the intervention group, respectively, and the difference was statistically significant (P0.05). Compared with the model group, the intervention group decreased the difference of 6.63% (P0.05).
The changes of Calpain activity in the 4 rats were increased by 33.33% (P0.01) in the model group and 15.87% (P0.01) in the intervention group. The Calpain activity of the spinal cord in the intervention group was 13.10% (P0.05), compared with the model group.
Compared with the control group, the changes of spinal cord neurofilament protein in the 5 rats were 30%, 60.9% (P0.05) and 14.1% and 25.9% (P0.05) in the intervention group, NF-L and NF-M respectively. Compared with the model group, the NF-L in the intervention group was 12.6% and 21.7% (P0.05), respectively.
conclusion
1ACR can induce neurobehavioral dysfunction in rats, and CP can antagonize abnormal changes.
2ACR caused the concentration of Ca2+ in the spinal cord, the activity of Calpain and the expression of NFs. CP inhibited the concentration of Ca2+, the activity of Calpain, and the increase of NFs, suggesting that CP had a neuroprotective effect on the rats infected with ACR.
3CP can reduce the accumulation of NFs induced by ACR, suggesting that the mechanism of ACR neurotoxicity may be related to the change of calpain.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965

【参考文献】