本文選題：氯胺酮 + GABAB受體　； 參考：《河北醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的：糖尿病神經(jīng)痛（diabetic neuropathy pain, DNP）是糖尿病常見(jiàn)的并發(fā)癥之一，在糖尿病患者中的發(fā)病率約為7.5%-24%，且其發(fā)病率呈逐年上升的趨勢(shì)，嚴(yán)重影響人們的生活質(zhì)量和心理健康。抑郁作為一種常見(jiàn)的情緒障礙與慢性疼痛密切相關(guān)。目前，大約12.7%的糖尿病神經(jīng)痛患者合并抑郁，但臨床尚缺乏有效的治療藥物及措施。氯胺酮作為一種非競(jìng)爭(zhēng)性的NMDA（N-甲基-D-天冬氨酸）受體拮抗劑，不僅具有顯著的鎮(zhèn)痛效果，近年來(lái)也發(fā)現(xiàn)其具有快速持久的抗抑郁作用，但其鎮(zhèn)痛抗抑郁的具體機(jī)制尚不清楚，因此本研究通過(guò)其機(jī)制探討為臨床治療提供理論依據(jù)。 研究表明：丘腦、海馬、大腦皮層等區(qū)域的GABAB（γ-氨基丁酸）受體蛋白表達(dá)下調(diào)在抑郁、焦慮、成癮等的形成過(guò)程中發(fā)揮著重要的作用，并且發(fā)現(xiàn)糖尿病神經(jīng)痛大鼠脊髓GABAB受體表達(dá)下調(diào)在疼痛的形成和維持中發(fā)揮重要作用。另有研究發(fā)現(xiàn)：在疼痛合并抑郁時(shí)，海馬、丘腦、大腦皮層等區(qū)域神經(jīng)元釋放腦源性神經(jīng)生長(zhǎng)因子（BDNF）明顯減少，給予鎮(zhèn)痛抗抑郁治療后，BDNF表達(dá)升高直至恢復(fù)正常，但在應(yīng)用氯胺酮鎮(zhèn)痛抗抑郁的過(guò)程中海馬GABAB受體及BDNF表達(dá)的變化目前尚不清楚。本實(shí)驗(yàn)通過(guò)腹腔注射鏈脲佐菌素（STZ），制備糖尿病神經(jīng)痛大鼠模型，應(yīng)用強(qiáng)迫游泳實(shí)驗(yàn)制備并評(píng)價(jià)抑郁模型，糖尿病神經(jīng)痛合并抑郁模型制備成功后，給予氯胺酮及NMDA受體激動(dòng)劑NMDA進(jìn)行干預(yù)。實(shí)驗(yàn)分為：正常對(duì)照組（C組）、糖尿病神經(jīng)痛合并抑郁模型（D1組）、氯胺酮組（D2組）、NMDA組（D3組）、氯胺酮+NMDA組（D4組），通過(guò)觀察不同組別大鼠50%機(jī)械縮足閾值（PWT）、強(qiáng)迫游泳實(shí)驗(yàn)不動(dòng)時(shí)間（IMSFT）變化，采用免疫組織化學(xué)染色法、RT-PCR法及Western blot法，比較各組大鼠海馬GABAB受體及BDNF的表達(dá)變化，從而探討氯胺酮鎮(zhèn)痛抗抑郁作用的可能機(jī)制。 方法：健康成年雄性SD大鼠30只，體重180～200g，由河北醫(yī)科大學(xué)實(shí)驗(yàn)動(dòng)物中心提供，隨機(jī)分為兩組：正常對(duì)照組（C組）和糖尿病神經(jīng)痛模型組（D組），腹腔分別注射生理鹽水或鏈脲佐菌素(STZ，60mg/kg)。20只大鼠腹腔注射STZ兩周后，有16只大鼠的空腹血糖16.7mmol/L，為糖尿病模型制備成功，然后對(duì)糖尿病大鼠進(jìn)行15min強(qiáng)迫游泳實(shí)驗(yàn)（每周1次）制備抑郁模型，方法為：將大鼠置于高40cm，直徑20cm圓柱形透明水缸中，水深25±5cm，水溫25±2℃，水面距缸上緣10±5cm，水深以大鼠后爪剛可觸及缸底卻不足以支撐身體為宜，游泳15min。腹腔注射STZ三周后，用von Frey Hairs測(cè)定機(jī)械縮足閾值，計(jì)算50%機(jī)械縮足閾值小于4g為糖尿病神經(jīng)痛大鼠模型制備成功(D組)。將正常對(duì)照組(C組)和實(shí)驗(yàn)組（D組）大鼠，通過(guò)5min強(qiáng)迫游泳實(shí)驗(yàn)記錄其不動(dòng)時(shí)間（immobility time of forced swimming test, IMFST：大鼠在水中停止掙扎、或顯示漂浮狀態(tài)、或僅作一些必要的輕微動(dòng)作保持頭部浮在水面上的時(shí)間視為不動(dòng)時(shí)間）。每只大鼠進(jìn)行強(qiáng)迫游泳實(shí)驗(yàn)之前更換玻璃缸內(nèi)的水，洗凈水缸，避免相互影響。與C組大鼠相比，IMFST顯著延長(zhǎng)者提示DNP合并抑郁模型可能制備成功。為驗(yàn)證模型制備成功，在行為學(xué)（PWT及IMFST）測(cè)定結(jié)束后，取C組和D組大鼠海馬組織，應(yīng)用免疫組化法測(cè)定BDNF陽(yáng)性細(xì)胞數(shù)，分子生物學(xué)方法測(cè)定其mRNA和蛋白表達(dá)變化。結(jié)合行為學(xué)及分子生物學(xué)變化，確定DNP合并抑郁大鼠的行為學(xué)條件，用于下一步成功制備DNP合并抑郁大鼠模型。 根據(jù)上一步實(shí)驗(yàn)結(jié)果確定的模型制備成功條件，制備DNP合并抑郁模型大鼠80只。根據(jù)給藥不同隨機(jī)分為四組（n=20）：模型組（D1組）：生理鹽水（0.5ml）+生理鹽水（0.5ml）；氯胺酮組（D2組）：氯胺酮10mg/kg(0.5ml)+生理鹽水(0.5ml)；NMDA組（D3）：生理鹽水(0.5ml)+NMDA15mg/kg(0.5ml)；氯胺酮+NMDA組（D4組）：氯胺酮10mg/kg(0.5ml)+NMDA15mg/kg(0.5ml)。連續(xù)肌肉注射給藥一周，，每天上午給藥。給藥完成后第1天(T1)、給藥完成后兩周（T2）分別測(cè)定大鼠PWT及IMFST，并于T1、T2時(shí)間點(diǎn)行為學(xué)測(cè)試后每組分別取10只大鼠海馬組織，采用免疫組化法和分子生物學(xué)技術(shù)測(cè)定大鼠海馬GABAB受體及BDNF表達(dá)變化。 結(jié)果：1腹腔注射STZ2周末，20只大鼠中有16只空腹血糖16.7mmol/L，為糖尿病模型制備成功。上述16只大鼠經(jīng)過(guò)1次15min的強(qiáng)迫游泳實(shí)驗(yàn)，于STZ注射3周末測(cè)定PWT及IMFST，其中10只大鼠PWT＜4g為大鼠DNP模型成功，且其IMFST均＞150s，其余大鼠剔除實(shí)驗(yàn)。與正常大鼠相比，10只DNP組大鼠PWT明顯降低, IMFST明顯延長(zhǎng)，其海馬組織BDNF陽(yáng)性細(xì)胞數(shù)明顯減少；mRNA含量明顯下降；蛋白表達(dá)顯著下降（P0.05）。因此，當(dāng)行為學(xué)測(cè)定糖尿病大鼠PWT＜4g且IMFST＞150s時(shí)，其海馬組織BDNF表達(dá)明顯下降，表明DNP合并抑郁模型制備成功。2與C組比較，D組大鼠各時(shí)間點(diǎn)PWT降低（P0.05），IMFST延長(zhǎng)（P0.05），BDNF陽(yáng)性細(xì)胞數(shù)減少（P0.05），mRNA及蛋白表達(dá)下降（P0.05），上述結(jié)果與模型大鼠變化一致，表明實(shí)驗(yàn)?zāi)Ｐ椭苽涑晒�，同時(shí)GABAB受體蛋白、mRNA表達(dá)以及陽(yáng)性細(xì)胞數(shù)均減少（P0.05）；與D1組比較，D2組大鼠海馬BDNF及GABAB受體的蛋白及mRNA表達(dá)均增加，陽(yáng)性細(xì)胞表達(dá)數(shù)也明顯上升（P0.05）；而D3、D4組大鼠海馬GABAB受體、BDNF的蛋白、mRNA表達(dá)以及陽(yáng)性細(xì)胞數(shù)均未見(jiàn)明顯變化（P0.05）。上述指標(biāo)各組T1與T2時(shí)間點(diǎn)相比均無(wú)明顯變化（P＞0.05）。 結(jié)論：氯胺酮可通過(guò)上調(diào)糖尿病神經(jīng)痛合并抑郁大鼠海馬GABAB受體及BDNF的表達(dá)，改善大鼠疼痛及抑郁狀態(tài)。
[Abstract]:Objective: diabetic neuropathy pain (DNP) is one of the common complications of diabetes. The incidence of diabetes is about 7.5%-24%, and its incidence is increasing year by year, which seriously affects people's quality of life and mental health. Depression as a common emotional disorder is closely related to chronic pain. At present, about 12.7% of patients with diabetic neuropathic pain are associated with depression, but there are still lack of effective therapeutic drugs and measures. Ketamine, as a non competitive NMDA (N- methyl -D- aspartic acid) receptor antagonist, not only has significant analgesic effect, but has also found its rapid and persistent antidepressant effect in recent years, but its analgesic and inhibitory effect The mechanism of depression is not clear. Therefore, this study will provide theoretical evidence for clinical treatment through its mechanism.
The study shows that the downregulation of GABAB (gamma aminobutyric acid) receptor protein expression in the thalamus, hippocampus and cerebral cortex plays an important role in the formation of depression, anxiety and addiction, and it is found that the down regulation of GABAB receptor expression in the spinal cord of diabetic rats plays an important role in the formation and maintenance of pain and pain. Now: the release of brain derived Neurogrowth factor (BDNF) in hippocampus, thalamus, cerebral cortex and other regions in pain and depression is significantly reduced. After the treatment of analgesic antidepressant treatment, the expression of BDNF increases until normal, but the changes in the expression of GABAB receptor and BDNF in hippocampus are not yet clear during the application of ketamine analgesic and antidepressant. The rat model of diabetic neuropathic pain was prepared by intraperitoneal injection of streptozotocin (STZ). Forced swimming test was used to prepare and evaluate the depression model. After the diabetic neuropain combined with depression model was successfully prepared, ketamine and NMDA receptor agonist NMDA were given. The experiment was divided into normal control group (C group) and diabetic neuralgia. Combined depression model (group D1), ketamine group (group D2), group NMDA (group D3) and group +NMDA (group D4) of ketamine (group D4), by observing the threshold of 50% mechanical contraction (PWT) in different groups of rats (PWT), the change of the time (IMSFT) of forced swimming test (IMSFT), immunohistochemistry, RT-PCR method and Western blot method were used to compare the hippocampal GABAB receptors and the tables of the rats in each group. To explore the possible mechanism of ketamine analgesia and antidepressant effect.
Methods: 30 healthy adult male SD rats, weighing 180 to 200g, were provided by the experimental animal center of Hebei Medical University. They were randomly divided into two groups: normal control group (group C) and diabetic neuropain model group (group D), peritoneal injection of saline or streptozotocin (STZ, 60mg/kg).20 in the abdominal cavity for two weeks, and 16 rats in the abdominal cavity. The fasting blood glucose 16.7mmol/L was successfully prepared for the diabetic model. Then the 15min forced swimming test (1 times a week) was used to prepare the depressive model. The method was to put the rats in a high 40cm, diameter 20cm cylindrical transparent cylinder, the water depth was 25 + 5cm, the water temperature was 25 + 2 C, the water surface was 10 + 5cm from the upper edge of the cylinder, and the water depth was just a palpable cylinder in the hind paw of the rat. The bottom was not sufficient to support the body. After three weeks of intraperitoneal injection of 15min. for swimming STZ, the threshold of mechanical contraction was measured with von Frey Hairs, and a successful model of diabetic neuropathic rat model was successfully prepared (group D) with the threshold of 50% mechanical shrinkage less than 4G. (group C) and experimental group (D group), the time of action was recorded by 5min forced swimming test (group D). Immobility time of forced swimming test, IMFST: the rats in the water stop struggling, or display the floating state, or only make some necessary minor movements to keep the head floating on the surface of the water as immobile). Each rat can replace the water in the glass cylinder before the forced swimming test, clean the water cylinder, avoid mutual influence. And C group. In order to verify the success of the DNP combined depression model, the IMFST significant prolongation suggested that the model was successful. In order to verify the success of the model preparation, the hippocampal tissues of the C and D groups were taken after the determination of the behavioral (PWT and IMFST). The number of BDNF positive cells was measured by immunohistochemical method, and the molecular biology method was used to determine the changes of mRNA and protein expression. And molecular biological changes, determine the behavioral conditions of DNP combined with depression rats, and prepare for the next successful preparation of DNP combined with depression rat model.
According to the successful condition of the model preparation determined by the results of the previous experiment, 80 rats with DNP combined with depression were prepared. According to the different dosage, the model group was randomly divided into four groups (n=20): the model group (group D1): physiological saline (0.5ml) + physiological saline (0.5ml); ketamine group (D2 group): chloramines 10mg/kg (0.5ml) + physiological saline (0.5ml); NMDA group (D3): physiological salt Water (0.5ml) +NMDA15mg/kg (0.5ml), ketamine +NMDA group (D4 group): Ketamine 10mg/kg (0.5ml) +NMDA15mg/kg (0.5ml). Continuous intramuscular injection for one week, every morning, after first days (T1). After the administration was completed, the rats were measured respectively, and 10 rats in each group were taken after the time point behavior test. The expression of GABAB receptor and BDNF in hippocampus of rats were determined by immunohistochemistry and molecular biology techniques.
Results: 1 on the weekend of intraperitoneal injection of STZ2, 20 rats had 16 fasting blood glucose 16.7mmol/L, which were successfully prepared for diabetes model. After 1 times of 15min forced swimming test, 16 rats were tested for PWT and IMFST at the end of STZ injection, of which 10 rats PWT < 4G as the DNP model of rats, and the IMFST > 150s, the rest of the rats were eliminated. Compared with normal rats, the PWT of 10 rats in the 10 group was obviously decreased, the IMFST was obviously prolonged, the number of BDNF positive cells in the hippocampus decreased obviously, the content of mRNA decreased obviously and the protein expression decreased significantly (P0.05). Therefore, the BDNF expression of the hippocampus was obviously decreased when the behavioral test of diabetic rats PWT < 4G and IMFST > 150s. Compared with group C, PWT decreased (P0.05), IMFST prolonged (P0.05), BDNF positive cells decreased (P0.05), mRNA and protein expression decreased (P0.05) at all time points in group D, and the above results were consistent with the model rats, indicating that the experimental model was successfully prepared, while the GABAB protein, the expression of positive cells and the number of positive cells were reduced. Less (P0.05); compared with group D1, the protein and mRNA expression of BDNF and GABAB receptors in hippocampus of group D2 increased, and the expression of positive cells also increased significantly (P0.05), while D3, BDNF protein, mRNA expression and number of positive cells were not obviously changed. Significant changes (P > 0.05).
Conclusion: ketamine can improve pain and depression in rats by up regulating the expression of GABAB receptor and BDNF in diabetic rats with neuropathic pain and depression.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R965

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 胡益民;程慧嫻;崔耀梅;高志勤;余海鷹;楊春;楊春;楊建軍;;氯胺酮對(duì)強(qiáng)迫游泳大鼠海馬谷氨酸、NR2A及NR2B的影響[J];解放軍醫(yī)學(xué)雜志;2012年05期

2 谷朝陽(yáng);安書(shū)成;;大鼠眶額葉GABA及其B型受體在應(yīng)激性抑郁行為發(fā)生中的作用及其影響機(jī)制[J];動(dòng)物學(xué)研究;2011年03期

3 鄧華聰;葛倩;劉金波;李丙蓉;程偉;;2型糖尿病患者胰島素受體酪氨酸激酶活性變化機(jī)制的探討[J];中華內(nèi)分泌代謝雜志;2006年01期

4 陸勤,陳忠,朱劍琴;GABA 對(duì)胚胎小鼠大腦皮質(zhì)神經(jīng)細(xì)胞生長(zhǎng)的影響及其作用機(jī)制[J];中國(guó)應(yīng)用生理學(xué)雜志;1997年02期

5 趙曉南;段衛(wèi)東;吳川;劉朋;劉飛飛;王秀麗;;硫化氫誘發(fā)疼痛大鼠脊髓背角N-甲基-D-天冬氨酸受體2B亞基的表達(dá)變化[J];中華實(shí)驗(yàn)外科雜志;2013年03期

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