本文選題：氧化應(yīng)激 + 糖尿病��； 參考：《南京醫(yī)科大學(xué)》2015年碩士論文

【摘要】：目的:1、探討糖尿病模型狀態(tài)下,氧化應(yīng)激對(duì)血循環(huán)及神經(jīng)組織中Neuritin水平的影響。2、研究血循環(huán)及神經(jīng)組織中Neuritin水平與神經(jīng)功能的可能關(guān)系。方法:1分組、造模及給藥雄性SD大鼠適應(yīng)性喂養(yǎng)1周,隨機(jī)選擇10只為正常對(duì)照組,其余大鼠為實(shí)驗(yàn)組。實(shí)驗(yàn)組大鼠禁食不禁水12h后,予一次性腹腔注射鏈尿佐菌素(STZ)55mg/kg,72h后剪尾取血,測血糖16.7mmol/L視為糖尿病造模成功,隨后隨機(jī)分為糖尿病組、低劑量硫辛酸、高劑量硫辛酸組,每組10只。硫辛酸組按20mg?kg-1?d-1或40mg?kg-1?d-1腹腔注射;糖尿病組予等量生理鹽水腹腔注射,實(shí)驗(yàn)期間自由攝食飲水,給藥2、4、6周。2坐骨神經(jīng)傳導(dǎo)速度(MNCV)測定參考Obrosova等[1]方法,分別在造模2、4、6周測定各組坐骨神經(jīng)MNCV。將大鼠用10%的水合氯醛350mg/kg腹腔注射麻醉后固定于實(shí)驗(yàn)平板,兩個(gè)刺激電極分別位于左側(cè)坐骨切跡、內(nèi)踝后方,記錄電極位于左足底部,單脈沖方波刺激,記錄兩個(gè)刺激點(diǎn)間的距離,輸入肌電圖儀,得出MNCV,重復(fù)三次取平均值。3尾神經(jīng)傳導(dǎo)速度(CNCV)測定刺激電極置于離尾部毛跡120mm處(遠(yuǎn)端),記錄電極置于離刺激電極100mm的軸向距離處(近端),參考電極分別置于離刺激電極近端和記錄電極遠(yuǎn)端約20mm處,刺激電極和記錄電極中間接地線.神經(jīng)反應(yīng)是由5 Hz頻率刺激引起的。直腸溫度保持在37℃。4神經(jīng)組織形態(tài)結(jié)構(gòu)觀察取一小段坐骨神經(jīng),用戊二醛液預(yù)固定,經(jīng)固定、包埋、常規(guī)制片,再用醋酸鈾和檸檬酸鉛雙染,最后用Tecnai透射電鏡觀察。5生化指標(biāo)檢測大鼠尾尖取血,FAD葡萄糖脫氫酶法測定血糖;大鼠眼眶靜脈取血,離心得到血清,硫代巴比妥酸法測定丙二醛(MDA)含量,羥胺法測定超氧化物歧化酶(SOD)活性,酶聯(lián)免疫吸附法測定neuritin濃度,均按試劑盒說明進(jìn)行檢測。6檢測坐骨神經(jīng)、背根神經(jīng)節(jié)neuritin蛋白表達(dá)水平提取組織總蛋白,BCA法測定蛋白質(zhì)濃度,取80μg蛋白實(shí)驗(yàn)。經(jīng)電泳、轉(zhuǎn)膜、封閉后,分別4℃敷育neuritin抗體和β-actin抗體過夜,再用相應(yīng)的HRP偶聯(lián)的二抗與其反應(yīng),加ECL化學(xué)發(fā)光試劑,采用Image Lab圖像分析軟件測定和比較各組neuritin蛋白相對(duì)表達(dá)量。7坐骨神經(jīng)和背根神經(jīng)節(jié)的鑒定兩種組織的蠟塊切成5微米的切片,常規(guī)步驟脫蠟。將切片用0.01M PBS處理進(jìn)行抗原修復(fù),用1%牛血清白蛋白的PBS封閉,接著用一抗和二體的稀釋封閉液孵育,細(xì)胞核可用4',6二脒基-2-苯基吲哚(DAPI)進(jìn)行染色。結(jié)果:1、糖尿病大鼠血糖明顯高于對(duì)照組,而糖尿病組與硫辛酸治療組間無明顯差異。2、與正常對(duì)照相比,糖尿病組2周后MDA濃度升高,SOD活力下降,neuritin的含量降低(P0.05),4周后MNCV開始下降(P0.05),坐骨神經(jīng)出現(xiàn)脫髓鞘病變;與糖尿病對(duì)照組相比,硫辛酸組MDA濃度下降,SOD活性升高,neuritin的含量增加,MNCV增加(P0.05)。3、正常組的坐骨神經(jīng)及背根神經(jīng)節(jié)中neuritin的表達(dá)高于糖尿病大鼠,2、4、6周的糖尿病大鼠neuritin的表達(dá)呈下降趨勢。結(jié)論:1、糖尿病大鼠中存在嚴(yán)重的氧化應(yīng)激和neuritin含量的下降,這些變化出現(xiàn)在外周神經(jīng)功能和結(jié)構(gòu)異常之前。2、硫辛酸可以改善氧化應(yīng)激,且成一定的量效關(guān)系。3、氧化應(yīng)激可能直接或者通過下調(diào)neuritin表達(dá)的相關(guān)機(jī)制引起糖尿病外周神經(jīng)的異常。
[Abstract]:Objective: 1, to investigate the effect of oxidative stress on the blood circulation and the Neuritin level in the nerve tissue in the state of diabetes mellitus (.2), and to study the possible relationship between the level of Neuritin and the nerve function in the blood circulation and nerve tissue. Methods: 1 groups, the model and the male SD rats were fed for 1 weeks, and 10 rats were randomly selected as the normal control group, and the other rats were randomly selected. For the experimental group, the rats in the experimental group were fasted for 12h, given a one-time intraperitoneal injection of streptozotocin (STZ) 55mg/kg, 72h after cutting the tail and taking the blood, and measuring blood sugar 16.7mmol/L as a successful model of diabetes, then randomly divided into diabetes group, low dose lipoic acid, high dose of lipoic acid group, 10 rats in each group, 20mg kg-1? D-1 or 40mg? Kg-1? D-1 abdomen in the group of lipoic acid. Intraperitoneal injection of the diabetes group was injected with equal amount of normal saline. During the experiment, drinking water was free, and the [1] method of.2 sciatic nerve conduction velocity (MNCV) for 2,4,6 weeks was measured, and the [1] method of Obrosova was measured at 2,4,6 weeks in the model, and the rats in each group of sciatic nerve MNCV. were injected with 10% chloral chloral 350mg/kg in the abdominal cavity and fixed on the experimental plate after the intraperitoneal injection of 10% chloral chloral 350mg/kg. The two stimulation electrodes were located at the left isch incisor and the posterior medial malleolus respectively. The recording electrode was located at the bottom of the left foot, stimulated by single pulse square wave, and recorded the distance between the two stimuli. The electromyogram was entered, and MNCV was obtained, and the three times of the average.3 tail nerve conduction velocity (CNCV) was placed at 120mm (Yuan Duan) and recorded at the tail of the tail. The axial distance (proximal) was placed at the axial distance from the stimulation electrode 100mm. The reference electrodes were placed at the proximal end of the electrode and the distal end of the recording electrode at about 20mm. The stimulation electrode and the recording electrode were grounded. The nerve reaction was caused by the 5 Hz frequency stimulation. The rectal temperature kept a small segment of the sciatic God at the morphological structure of the.4 nerve tissue at 37 degrees centigrade. It was prefixed with glutaraldehyde solution, immobilized, embedded, conventional and double stained with uranium acetate and lead citrate. At the end, blood was detected by.5 biochemical indexes by Tecnai transmission electron microscopy, and blood glucose was measured by FAD glucose dehydrogenase, blood from orbital veins of rats, centrifugation, and determination of malondialdehyde (MDA) by thiobarbituric acid method. The activity of superoxide dismutase (SOD) was measured by hydroxylamine method and the concentration of Neuritin was measured by enzyme linked immunosorbent assay (ELISA). All the sciatic nerves were detected by.6 in the reagent box. The total protein of Neuritin protein in the dorsal root ganglion was extracted. The protein concentration was measured by BCA method and 80 micron protein experiment was taken. After electrophoresis, membrane transfer and closure, it was applied at 4 C respectively. Neuritin antibody and beta -actin antibody were overnight, and the corresponding HRP coupled two antibodies were reacted with ECL chemiluminescence reagents. The Image Lab image analysis software was used to determine and compare the relative expression of Neuritin protein in each group.7 sciatic nerve and dorsal root ganglion. The wax blocks of the two tissues were cut into 5 microns, and the conventional steps were dewaxed. The slice was treated with 0.01M PBS for antigen repair, closed with PBS of 1% bovine serum albumin, and incubated with one and two body diluent closed liquid. The nucleus could be stained with 4', 6 two amid -2- phenyl indole (DAPI). Results: 1, diabetic rats were significantly higher than the control group, and there was no significant difference between the diabetic group and the lipoic acid group.2, Compared with the normal control, the concentration of MDA increased after 2 weeks, the activity of SOD decreased and the content of Neuritin decreased (P0.05). After 4 weeks, MNCV began to decrease (P0.05), and the sciatic nerve appeared demyelinating disease. Compared with the diabetes control group, the concentration of MDA in the lipoic acid group decreased, the SOD activity increased, the Neuritin content increased, MNCV increased (P0.05).3, normal group. The expression of Neuritin in the sciatic and dorsal root ganglia was higher than that in diabetic rats. The expression of Neuritin in diabetic rats was decreased in 2,4,6 weeks. Conclusion: 1, there are severe oxidative stress and a decrease in Neuritin content in diabetic rats. These changes appear before peripheral nerve function and structural abnormalities.2, and lipoic acid can improve oxygen. Oxidative stress and a certain dose effect relationship.3, oxidative stress may directly or through the down-regulation of Neuritin expression mechanism related to diabetic peripheral nerve abnormalities.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R587.2

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2 武小希;氧化應(yīng)激誘導(dǎo)人臍靜脈內(nèi)皮細(xì)胞microRNA表達(dá)譜的改變及驗(yàn)證[D];河北醫(yī)科大學(xué);2015年

3 張文松;Nrf2/HO-1通路在氧化應(yīng)激致終末期腎病血管鈣化中的作用及機(jī)制研究[D];川北醫(yī)學(xué)院;2015年

4 王倩;阿托伐他汀治療急性缺血性腦卒中的臨床研究[D];河北醫(yī)科大學(xué);2015年

5 李盼盼;活性羰基化合物誘導(dǎo)的氧化應(yīng)激致細(xì)胞能量代謝障礙和毒性的機(jī)制[D];西北農(nóng)林科技大學(xué);2015年

6 安美玲;CCK-8對(duì)甲基苯丙胺致神經(jīng)損傷的保護(hù)作用及氧化應(yīng)激機(jī)制研究[D];河北醫(yī)科大學(xué);2015年

7 劉金蓮;氧化應(yīng)激在動(dòng)靜脈內(nèi)瘺血栓形成中的作用[D];延邊大學(xué);2015年

8 王琦;白藜蘆醇抑制肥胖相關(guān)的脂肪組織炎癥與氧化應(yīng)激及對(duì)睪丸內(nèi)分泌功能的保護(hù)[D];安徽醫(yī)科大學(xué);2015年

9 關(guān)瑾;體外研究羧基化多壁碳納米管誘發(fā)的氧化應(yīng)激效應(yīng)與機(jī)理[D];山東大學(xué);2015年

10 張斌;體外實(shí)驗(yàn)研究不同粒徑納米銀的氧化應(yīng)激效應(yīng)[D];山東大學(xué);2015年

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