本文選題：丙烯酰胺 + 鈣蛋白酶抑制劑��； 參考：《山東大學》2014年碩士論文

【摘要】：目的 丙烯酰胺(acrylamide, ACR)應用廣泛,以它為單體作為原料合成的工業(yè)產(chǎn)品多達百種。ACR能誘發(fā)中樞-周圍神經(jīng)病。臨床表現(xiàn)有出汗、手腳感覺異常、腿部痙攣、白指、皮膚刺激和皮膚剝落等,之后表現(xiàn)為骨骼肌無力和四肢麻痹。ACR誘導的病變尚無定論,國內(nèi)外研究多集中于神經(jīng)元軸索,但是近年來國內(nèi)外學者研究表明ACR誘導的細胞Ca2+內(nèi)流,鈣依賴性蛋白酶活性升高,病理特征顯示軸突內(nèi)腫脹和神經(jīng)絲堆積。本研究之前以建立ACR中毒模型,發(fā)現(xiàn)組織內(nèi)的神經(jīng)絲(neurofilaments, NFs)發(fā)生了明顯改變。但其中NFs是如何改變的尚不清楚。我們假設這種NFs的改變與Calpain有關,為驗證這種假設,本研究通過建立ACR中毒神經(jīng)病大鼠模型及鈣蛋白酶抑制劑(Calpeptin, CP)干預模型,測定大鼠神經(jīng)行為學的變化及脊髓組織中Ca2+、Calpain活性、NFs蛋白表達水平,為研究ACR引起的神經(jīng)病變的發(fā)病機制和預防措施提供理論依據(jù)。 方法 1動物模型及行為觀察將30只成年雌性Wistar大鼠適應性飼養(yǎng)一周后,隨機分為3組,即對照組、模型組和干預組(n=10)。ACR溶解生理鹽水中,模型組和干預組按30mg/kg·bw經(jīng)腹腔注射染毒,對照組給予等體積生理鹽水。干預組在注射ACR1h后,給予Calpeptin200μg/kg-bw,每3次／周,連續(xù)4周。于第4周,斷頭處死大鼠,取大腦、脊髓備用。每周測體重及神經(jīng)行為學指標1次。 2神經(jīng)行為學指標測定給抑制劑期間測定不同時間點各組大鼠神經(jīng)行為學指標：步態(tài)評分、后肢撐力指數(shù)。 3分離脊髓,胰酶消化后,Fura-2/AM裝載后用熒光分光光度計測定鈣離子含量。 4取脊髓經(jīng)組織勻漿上清,加Calpain substrate II,用酶標儀測定熒光強度,得出Calpain活性。 5蛋白免疫印跡(Western blotting)測定脊髓組織中的NF-L、NF-M蛋白表達水平。 結果 1大鼠一般狀況變化對照組大鼠體重平穩(wěn)增長；模型組大鼠體重增長較為緩慢,兩者體重于第2周開始出現(xiàn)顯著差異(P0.05)；干預組大鼠體重在第2周時體重顯著高于模型組(P0.05)。 2大鼠神經(jīng)行為學改變模型組大鼠腹部著地,兩腿叉開平鋪、劃行、呈鴨步,干預組出現(xiàn)不同程度的步態(tài)異常,后肢間距增寬,但異常程度較模型組輕,其中模型組步態(tài)評分呈3分的占60%,干預組2-3分大鼠分別占80%和20%,與模型組比差異有統(tǒng)計學意義(P0.01)。后肢撐力實驗結果顯示,模型組大鼠后肢間距比對照組明顯增寬(P0.01),干預組大鼠后肢間距明顯比模型組降低,到第4周,干預組大鼠后肢間距比模型組降低,差異有統(tǒng)計學意義P0.05)。 3大鼠脊髓組織Ca2+變化與對照組相比,模型組和干預組分別升高了25.34%和17.03%,差異有統(tǒng)計學意義(P0.05)；與模型組比較,干預組降低了6.63%差異有統(tǒng)計學意義(P0.05)。 4大鼠Calpain活性的改變與對照組比,模型組脊髓Calpain活性升高了33.33%(P0.01),干預組升高了15.87%(P0.01)；與模型組比,干預組脊髓Calpain活性降低了13.10%(P0.05)。 5大鼠脊髓神經(jīng)絲蛋白改變與對照組相比,模型組NF-L、NF-M分別升高了30.0%、60.9%(P0.05),干預組NF-L、NF-M分別升高了14.1%和25.9%(P0.05)。與模型組相比,干預組的NF-L、NF-M分別下降了12.6%和21.7%(P0.05)。 結論 1ACR可引起大鼠的神經(jīng)行為功能異常, CP能拮抗其異常改變。 2ACR引起脊髓中Ca2+濃度、Calpain活性、NFs表達增高,CP抑制了Ca2+濃度、Calpain活性、NFs的升高,提示CP對ACR染毒大鼠具有神經(jīng)保護作用。 3CP能降低ACR引起的NFs的堆積程度,提示ACR的神經(jīng)毒性作用機制可能與calpain的變化相關
[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.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R965

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