本文選題：川芎嗪 + 帕金森氏病　； 參考：《第四軍醫(yī)大學(xué)》2015年碩士論文

【摘要】：目的:帕金森病(Parkinson’S disease PD)是一種較為常見(jiàn)的椎體外系變性性疾病,由于其確切的發(fā)病機(jī)制迄今尚不十分明確,仍沒(méi)有根治的辦法。臨床上多選用藥物治療延緩病情惡化,但由于長(zhǎng)期用藥導(dǎo)致療效降低、毒副作用不耐受等,使患者不得不減少劑量或終止藥物治療。因此,尋找治療效果好、安全性高的PD藥物是當(dāng)前的研究熱點(diǎn)。川芎嗪(Tetramethylpyrazine,TMP)是從中藥川芎中提取的有效成分,其被廣泛應(yīng)用于治療神經(jīng)和心血管性疾病。近年來(lái),一些研究已經(jīng)表明,川芎嗪在體內(nèi)外具有神經(jīng)保護(hù)作用。川芎嗪也被發(fā)現(xiàn)具有缺血性腦損傷的保護(hù)作用,并可促進(jìn)細(xì)胞增殖與分化。1-甲基-4-苯基-1,2,3,6-四氫吡啶(MPTP)是一種神經(jīng)毒素,對(duì)參與PD發(fā)生的神經(jīng)元具有靶向作用。因此,MPTP已被廣泛應(yīng)用于帕金森病動(dòng)物模型的建立。本實(shí)驗(yàn)的目的是探討川芎嗪對(duì)MPTP所致大鼠中腦多巴胺神經(jīng)元損傷的保護(hù)作用及可能的抗凋亡抗氧化機(jī)制,為帕金森氏病的臨床治療提供分子基礎(chǔ)。方法:1.動(dòng)物分組與模型處理大鼠隨機(jī)分為4組,每組10只,分別為:對(duì)照組(Con,生理鹽水注射),MPTP組(MPTP與生理鹽水混合注射),TMP組(TMP注射),TMP+MPTP組(TMP與MPTP混合注射)。TMP(TMP 20mg/kg/d,TMP溶解于3%DMSO中,腹腔注射)給藥7天。在第7天,大鼠黑質(zhì)(SN)注入MPTP(1μmol MPTP溶解于2μl生理鹽水中,參照坐標(biāo):AP:-5.0 mm,ML:±2.1 mm,DV:-7.7 mm,使用30量規(guī)針頭從大鼠前鹵,中線和頭骨表面以0.35μl/min的速度注入),隨后繼續(xù)TMP給藥7天。步行階梯實(shí)驗(yàn)在第14天的14:00-18:00經(jīng)行。實(shí)驗(yàn)后,將大鼠處死并解剖,取出紋狀體和黑質(zhì)以備后續(xù)實(shí)驗(yàn)。2.步行階梯實(shí)驗(yàn)步行階梯實(shí)驗(yàn)用于評(píng)價(jià)大鼠前后肢體的協(xié)調(diào)能力。在測(cè)試前大鼠被訓(xùn)練3天進(jìn)行橫跨梯子的訓(xùn)練,記錄第14天進(jìn)行的3次測(cè)試的平均成績(jī)。3.多巴胺及其代謝產(chǎn)物的測(cè)定大鼠脫頸髓處死,冰臺(tái)上迅速斷頭將紋狀體取出,稱重,于-80℃儲(chǔ)存。檢測(cè)時(shí)加入含有DHBA內(nèi)標(biāo)的0.1mol/L的高氯酸溶液0.5ml,勻漿,于4℃低溫離心,15000r/min,離心l0min,取上清50μl用于高效液相色譜電化學(xué)檢測(cè)。4.TBARS和GSH的測(cè)定參照說(shuō)明書方法測(cè)定巴比土酸活性物質(zhì)(TBARS)和谷胱甘肽(GSH)含量5.免疫組化染色將處理后的大鼠腦組織-20℃冷凍并連續(xù)切片(5um),置于玻璃載玻片上,在4oC下細(xì)胞與特異性抗體共同孵育過(guò)夜,與Cy3抗體共同培養(yǎng)。然后置于共聚焦顯微鏡下觀察拍照。6.免疫組化檢測(cè)酪氨酸羥化酶(TH)陽(yáng)性細(xì)胞數(shù)造模一周后大鼠斷頭處死,在冰上取中腦腹側(cè),立即將其置于4%多聚甲醛中固定,蔗糖脫水后進(jìn)行冰凍切片,以15μm厚度連續(xù)切片,晾片后加大鼠抗TH單克隆一抗(1:1000),37℃下孵育l小時(shí),PBS洗3次每次5分鐘,加入熒光標(biāo)記的兔抗大鼠二抗,37℃孵育60分鐘,PBS洗3次,每次5分鐘經(jīng)封皮后熒光顯微鏡鏡下觀察黑質(zhì)致密部(SNc)的TH陽(yáng)性細(xì)胞數(shù),圖像分析系統(tǒng)計(jì)數(shù),每組取5個(gè)動(dòng)物,每個(gè)動(dòng)物取5張切片,計(jì)算平均數(shù)值。7.Western Blot實(shí)驗(yàn)檢測(cè)相關(guān)蛋白表達(dá)水平結(jié)果:1.川芎嗪對(duì)MPTP誘導(dǎo)的運(yùn)動(dòng)障礙的影響與Con組大鼠相比,MPTP組大鼠明顯花費(fèi)更多時(shí)間通過(guò)階梯,證實(shí)MPTP誘導(dǎo)大鼠產(chǎn)生運(yùn)動(dòng)障礙;與MPTP組相比,TMP+MPTP組大鼠通過(guò)階梯時(shí)間明顯縮短,結(jié)果表明,TMP的干預(yù),使MPTP誘導(dǎo)的大鼠運(yùn)動(dòng)障礙得到改善。2.川芎嗪對(duì)MPTP誘導(dǎo)的DA能神經(jīng)元損傷的影響與Con組相比,MPTP組大鼠酪氨酸羥化酶免疫反應(yīng)明顯減少,DA和DOPAC水平明顯降低;應(yīng)用TMP干預(yù)后,抑制了DA和DOPAC水平的降低,可顯著防止MPTP誘導(dǎo)的DA能神經(jīng)元損傷。3.TMP對(duì)MPTP誘導(dǎo)的細(xì)胞凋亡的影響為了評(píng)估細(xì)胞凋亡的相關(guān)改變,我們檢測(cè)Bax和Bcl-2的蛋白表達(dá)。在黑質(zhì)中,MPTP組大鼠Bax蛋白表達(dá)增加,Bcl-2蛋白表達(dá)降低。而MPTP誘導(dǎo)的Bax和Bcl-2的蛋白表達(dá)改變能被TMP有效抑制。另外,我們檢測(cè)了細(xì)胞質(zhì)中細(xì)胞色素c的釋放,與Con組相比,MPTP組細(xì)胞色素c釋放增多,而TMP干預(yù)后細(xì)胞色素c的釋放得到有效抑制。我們同時(shí)也檢測(cè)了capase3的剪切,MPTP可促進(jìn)capase3發(fā)生剪切,而TMP可顯著降低capase3的剪切。這些結(jié)果說(shuō)明TMP可抑制MPTP誘導(dǎo)的大鼠大腦黑質(zhì)細(xì)胞凋亡。4.TMP對(duì)MPTP誘導(dǎo)的誘導(dǎo)的氧化應(yīng)激的影響我們檢測(cè)了TMP和MPTP對(duì)大鼠大腦黑質(zhì)氧化還原平衡的影響。與Con組相比,MPTP組TBARS水平顯著增加;說(shuō)明MPTP引起了氧化損傷的發(fā)生。而TMP干預(yù)后可有效抑制TBARS水平的增高。MPTP組與Con組相比,GSH含量顯著降低。而TMP干預(yù)后可以有效抑制其降低。與Con組相比,MPTP組Nrf2和GCLc表達(dá)明顯降低降低。而TMP干預(yù)后能有效抑制Nrf2和GCLc的表達(dá)降低。這些結(jié)果說(shuō)明TMP可抑制MPTP誘導(dǎo)的大鼠大腦黑質(zhì)氧化應(yīng)激損傷。結(jié)論:1.川芎嗪可以改善MPTP誘導(dǎo)的大鼠運(yùn)動(dòng)障礙并可顯著防止MPTP誘導(dǎo)的多巴胺能神經(jīng)元損傷。2.川芎嗪的抗凋亡,抗氧化作用是其產(chǎn)生保護(hù)神經(jīng)元損傷作用的可能機(jī)制。3.川芎嗪的抗凋亡作用是通過(guò)其抑制Bax表達(dá),增加Bcl-2表達(dá),抑制細(xì)胞色素c的釋放,抑制caspase3的裂解來(lái)實(shí)現(xiàn)的;其抗氧化作用與調(diào)節(jié)Nrf2和GCLc有關(guān)。
[Abstract]:Objective: Parkinson's disease (Parkinson 'S disease PD) is a more common degenerative disease of the vertebral body. Because its exact pathogenesis is not yet very clear, there is still no cure for it. The people have to reduce the dose or terminate the drug treatment. Therefore, to find the effective and safe PD drugs is the current research hotspot. Ligustrazine (Tetramethylpyrazine, TMP) is an effective component extracted from Ligusticum chuanxiong, which is widely used in the treatment of nerve and heart blood tube diseases. In recent years, some studies have shown that Ligustrazine. Tetramethylpyrazine has also been found in the body and in vivo. Ligustrazine has also been found to have protective effects on ischemic brain damage, and can promote cell proliferation and differentiation,.1- methyl -4- phenyl -1,2,3,6- four hydropyridine (MPTP) is a neurotoxin that is targeted to neurons involved in PD. Therefore, MPTP has been widely used in the animal model of Parkinson's disease. The purpose of this experiment was to explore the protective effect of Ligustrazine on the injury of dopamine neurons in the mesencephalon of MPTP rats and the possible anti apoptosis antioxidation mechanism, and to provide the molecular basis for the clinical treatment of Parkinson's disease. Methods: 1. animal groups and model rats were randomly divided into 4 groups, 10 in each group, Con, Saline injection), group MPTP (MPTP and saline mixed injection), group TMP (TMP injection), TMP+MPTP group (TMP and MPTP mixed injection).TMP (TMP 20mg/kg/d, TMP dissolved in 3%DMSO, intraperitoneal) for 7 days. On the seventh day, the rat substantia nigra was injected into 2 mu saline. 7 mm, using the 30 gauge needle from the anterior brine of the rat, the middle line and the surface of the skull at the speed of 0.35 mu l/min, and then continue to TMP for 7 days. The walking ladder experiment was performed on the fourteenth day of 14:00-18:00. After the experiment, the rats were killed and dissected, the striatum and the substantia nigra were taken out for the follow-up test of the walking ladder experiment of the.2. walking ladder test used for evaluation. The rat's ability to coordinate the limbs before and after the test. The rats were trained for 3 days before the test. The average score of the 3 tests on fourteenth days was recorded,.3. dopamine and its metabolites were measured. The rats were removed from the cervix, and the striatum was removed, weighed and stored at -80 C. The DHBA internal standard was added to the test. 0.1mol/L's perchloric acid solution 0.5ml, homogenate, centrifugation at 4 centigrade, 15000r/min, centrifugation l0min, and supernatant 50 mu L for high performance liquid chromatography electrochemical detection.4.TBARS and GSH determination reference manual method for determination of barbiulic acid active substance (TBARS) and glutathione (GSH) content 5. immuno histochemical staining of brain tissue at -20 C after treatment Frozen and continuous slice (5um), placed on glass slides, incubated with specific antibodies in 4oC cells for the night, co culture with Cy3 antibody, and then placed under confocal microscope and observed and photographed.6. immunohistochemistry for the detection of tyrosine hydroxylase (TH) positive cells for a week, the rats were killed and the midbrain was taken on the ice, immediately on the ventral side of the brain. It was fixed in 4% polyformaldehyde. After the sucrose was dehydrated, the frozen section was sliced and sliced with 15 m thickness. After airing, the rat anti TH monoclonal antibody (1:1000) was increased, l hours were incubated at 37 degrees, PBS was washed for 3 times for 5 minutes. The rabbits were added to the fluorescent label for two anti rats, 37 centigrade was incubated for 60 minutes, the PBS was washed for 3 times, and the fluorescence microscopy after 5 minutes after 5 minutes was used. The number of TH positive cells in the dense part of the substantia nigra (SNc) was observed under the microscope, and the image analysis system was counted. 5 animals were taken in each group. 5 slices of each animal were taken, and the average value of.7.Western Blot was calculated to determine the expression level of related proteins: 1. the effect of Ligustrazine on the obstacle induced by MPTP was compared with that of the Con group, and the rats in the MPTP group were significantly spent. More time passed the ladder to confirm that MPTP induced the dyskinesia in rats; compared with the MPTP group, the rats in the group TMP+MPTP were significantly shortened through the ladder time. The results showed that the intervention of TMP, caused by MPTP induced dyskinesia, could improve the effect of.2. tetramethylpyrazine on the DA energy damage induced by MPTP, compared with the Con group, and the MPTP group rat tyrosine. The immunoreaction of hydroxylase was significantly reduced and the levels of DA and DOPAC were significantly reduced. The reduction of DA and DOPAC levels was inhibited by the use of TMP stem. The effects of MPTP induced DA neurons on the apoptosis induced by MPTP induced by.3.TMP were obviously prevented. We detected the protein expression of Bax and Bcl-2. In the substantia nigra, we detected the protein expression of Bax and Bcl-2 in the substantia nigra. The expression of Bax protein in the MPTP group increased and the expression of Bcl-2 protein decreased. The protein expression changes of Bax and Bcl-2 induced by MPTP could be effectively suppressed by TMP. In addition, we detected the release of cytochrome C in the cytoplasm. Compared with the Con group, the c release of cytochrome in the MPTP group increased, and the release of cytochrome MPTP was effectively suppressed after the TMP intervention. At the same time, the shear of capase3 was also detected, and MPTP could promote the shear of capase3, while TMP significantly reduced the shear of capase3. These results suggest that TMP can inhibit the effect of MPTP induced rat cerebral nigra apoptosis.4.TMP on the induced oxidative stress induced by MPTP, and we detected the redox balance of TMP and MPTP on rat brain black matter. Effect. Compared with group Con, the level of TBARS in group MPTP was significantly increased, indicating that MPTP caused oxidative damage, and TMP intervention could effectively inhibit the level of TBARS,.MPTP group was significantly lower in GSH content compared with Con group, while TMP intervention could effectively inhibit its decrease. The intervention can effectively inhibit the decrease of Nrf2 and GCLc expression. These results suggest that TMP can inhibit the oxidative stress injury in rat cerebral substantia nigra induced by MPTP. Conclusion: 1. Ligustrazine can improve the dyskinesia induced by MPTP and prevent the MPTP induced dopaminergic neurons to damage the apoptosis of.2. tetramethylpyrazine, and the antioxidant effect is its production. The possible mechanism for the protection of neuron damage,.3. tetramethylpyrazine's anti apoptosis effect is realized by inhibiting the expression of Bax, increasing the expression of Bcl-2, inhibiting the release of cytochrome c and inhibiting the cracking of Caspase3, and its antioxidant effect is related to the regulation of Nrf2 and GCLc.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R742.5

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 田學(xué)文,章翔,張劍寧;雌激素對(duì)帕金森病大鼠中腦黑質(zhì)多巴胺能神經(jīng)元保護(hù)作用的研究[J];濱州醫(yī)學(xué)院學(xué)報(bào);2004年03期

2 蔣知新,張清華,喻陸,衣志勇;咖啡因?qū)ε两鹕∩窠?jīng)保護(hù)作用的研究進(jìn)展[J];中華神經(jīng)醫(yī)學(xué)雜志;2004年05期

3 沈偉,孫圣剛;美滿霉素與帕金森病[J];國(guó)外醫(yī)學(xué).神經(jīng)病學(xué)神經(jīng)外科學(xué)分冊(cè);2004年06期

4 田有勇,孫圣剛;帕金森病神經(jīng)保護(hù)的實(shí)驗(yàn)研究進(jìn)展[J];國(guó)外醫(yī)學(xué).神經(jīng)病學(xué)神經(jīng)外科學(xué)分冊(cè);2005年02期

5 孫文虹,丁曉飛,郭麗萍,蔡穎,于多,倪小虎;川芎嗪促進(jìn)大鼠學(xué)習(xí)記憶作用的研究[J];哈爾濱商業(yè)大學(xué)學(xué)報(bào)(自然科學(xué)版);2004年01期

6 姚國(guó)恩,王景周,陳曼娥;川芎嗪對(duì)腦缺血后學(xué)習(xí)記憶障礙的療效評(píng)價(jià)[J];腦與神經(jīng)疾病雜志;2003年02期

7 邊艷珠,劉懷軍,郭艷蘇,周立霞,李金福,張文艷,田國(guó)章,魏強(qiáng);司來(lái)吉蘭對(duì)帕金森病小鼠黑質(zhì)的神經(jīng)保護(hù)作用及其評(píng)價(jià)[J];腦與神經(jīng)疾病雜志;2004年06期

8 王宗敏，徐正，王萬(wàn)鐵;家兔心肌缺血再灌注損傷及川芎嗪保護(hù)作用的超微結(jié)構(gòu)改變[J];溫州醫(yī)學(xué)院學(xué)報(bào);1998年01期

9 程曉馨,李豐橋,黃敏,王曉民,韓濟(jì)生;雷公藤氯內(nèi)酯醇對(duì)帕金森病大鼠多巴胺神經(jīng)元的保護(hù)作用[J];藥學(xué)學(xué)報(bào);2002年05期

10 劉衛(wèi)國(guó),陳生弟,陳琰,陸國(guó)強(qiáng),梁梁,徐潔懿;左旋多巴對(duì)C17.2神經(jīng)干細(xì)胞的毒性作用及司來(lái)吉蘭的神經(jīng)保護(hù)作用[J];中國(guó)神經(jīng)科學(xué)雜志;2004年01期

，

本文編號(hào)：2024905