本文選題：帕金森病 + JNK信號(hào)通路　； 參考：《北京中醫(yī)藥大學(xué)》2017年博士論文

【摘要】：目的:觀察針刀干預(yù)對(duì)帕金森病模型大鼠黑質(zhì)神經(jīng)炎癥、神經(jīng)細(xì)胞凋亡及相關(guān)因子的影響,并探討JNK信號(hào)通路在針刀干預(yù)帕金森病中的作用機(jī)制。方法:雄性SD大鼠72只,分為空白組、假手術(shù)組、模型組、藥物組、電針組和針刀組。采用單側(cè)紋狀體雙靶點(diǎn)注射6-OHDA建立PD模型,通過(guò)阿樸嗎啡(APO)誘發(fā)行為學(xué)檢測(cè)驗(yàn)證模型。空白組不做任何處理;假手術(shù)組與模型組的注射方法和部位相同,僅注射等量的含0.2%維生素C的生理鹽水;造模成功后,藥物組美多芭片50mg/kg灌胃,電針組進(jìn)行針刺治療,針刀組給予針刀干預(yù)。治療四周后各組取材檢測(cè),檢測(cè)指標(biāo)包括:(1)行為學(xué)檢測(cè):記錄大鼠60min內(nèi)旋轉(zhuǎn)圈數(shù);(2)HE染色觀察黑質(zhì)神經(jīng)細(xì)胞形態(tài)學(xué);(3)免疫熒光雙標(biāo)法檢測(cè)黑質(zhì)COX-2/TH,p-c-Jun/TH的表達(dá);(4)Westernblot檢測(cè)黑質(zhì)JNK,P-JNK,p-c-Jun,COX-2及TH表達(dá);(5)免疫組織化學(xué)檢測(cè)黑質(zhì)TH神經(jīng)細(xì)胞;(6)Real-TimePCR檢測(cè)黑質(zhì)DATmRNA含量;(7)TUNEL染色檢測(cè)凋亡細(xì)胞(8)免疫熒光雙標(biāo)法檢測(cè)黑質(zhì)Caspase-3/TH的表達(dá)(9)Westernblot檢測(cè)黑質(zhì)Caspase-3,Bcl-2,Beclin-1 的表達(dá)結(jié)果:(1)行為學(xué)檢測(cè):模型組出現(xiàn)旋轉(zhuǎn)行為且治療前后旋轉(zhuǎn)圈數(shù)無(wú)差異(P0.05);藥物組、電針組、針刀組干預(yù)后大鼠旋轉(zhuǎn)圈數(shù)明顯減少,活動(dòng)增多,干預(yù)前后有明顯差異(P0.01),與模型組相比差異顯著(P0.01)。(2)形態(tài)學(xué)檢測(cè):①HE染色:不同干預(yù)方法均可使HE染色黑質(zhì)神經(jīng)細(xì)胞數(shù)目增多,形態(tài)結(jié)構(gòu)趨于正常,藥物組黑質(zhì)神經(jīng)細(xì)胞排列有序,數(shù)量與模型組比較增多,細(xì)胞變性程度減輕,膠質(zhì)細(xì)胞增生不明顯;電針組黑質(zhì)神經(jīng)細(xì)胞數(shù)量與模型組比較增多較為明顯,細(xì)胞變性程度明顯減輕;針刀組黑質(zhì)神經(jīng)細(xì)胞數(shù)目與模型組比較明顯增多,排列較密集,細(xì)胞變性減輕。②黑質(zhì)TH的免疫組織化學(xué)染色:正常組和假手術(shù)組的黑質(zhì)均可見(jiàn)密集的TH免疫反應(yīng)陽(yáng)性細(xì)胞,呈棕褐色,胞體較大,胞體主要為圓形或卵圓形,少數(shù)為多邊形。模型組黑質(zhì)可見(jiàn)少量、稀疏的TH免疫反應(yīng)陽(yáng)性神經(jīng)元,胞體縮小,輪廓不清晰;藥物組、電針組及針刀組TH免疫反應(yīng)陽(yáng)性神經(jīng)元數(shù)量增多較為明顯,胞體輪廓清晰可見(jiàn),染色強(qiáng)度上亦有增加。③免疫熒光雙標(biāo)記黑質(zhì)COX-2/TH,p-c-Jun/TH,Caspase-3/TH檢測(cè)結(jié)果:正常組和假手術(shù)組的黑質(zhì)均可見(jiàn)密集的TH免疫反應(yīng)陽(yáng)性細(xì)胞,呈綠色,胞體較大,胞體主要為圓形或卵圓形,同時(shí),COX-2,p-c-Jun,Caspase-3/TH,呈紅色熒光,在數(shù)量上和染色強(qiáng)度上表達(dá)較弱;模型組黑質(zhì)可見(jiàn)少量、稀疏的TH免疫反應(yīng)陽(yáng)性神經(jīng)元,呈綠色,細(xì)胞腫脹變性,或皺縮,數(shù)量較少,染色強(qiáng)度減低,同時(shí),COX-2,p-c-Jun,Caspase-3,呈紅色熒光,在數(shù)量上和染色強(qiáng)度上表達(dá)增加;藥物組、電針組及針刀組TH免疫反應(yīng)陽(yáng)性神經(jīng)元呈綠色,排列較有序,數(shù)量增多,胞體變性程度減輕,染色強(qiáng)度上亦有增加;同時(shí),COX-2,p-c-Jun,Caspase-3/TH,呈紅色熒光,較模型組,在數(shù)量上減少,染色強(qiáng)度減低。與電針組和藥物組比較,針刀組TH神經(jīng)元數(shù)目更多,胞體變性程度更輕;針刀組COX-2和p-c-Jun在數(shù)量上較電針組少,但比藥物組多;藥物組Caspase-3在數(shù)量上比電針組和針刀組少。(3)Western blot 檢測(cè)結(jié)果:①與空白組比較,模型組大鼠黑質(zhì)內(nèi)JNK含量無(wú)明顯差異(P0.05);P-JNK,P-JNK/JNK,p-c-Jun表達(dá)升高(P0.05),COX-2表達(dá)顯著升高(P0.01);與模型組比較,藥物組、電針組、針刀組大鼠黑質(zhì)內(nèi)JNK含量無(wú)明顯差異(P0.05);與模型組比較,P-JNK和p-c-Jun含量在藥物組和電針組中有差異(P0.05),針刀組具有顯著差異(P0.01),與模型組比,P-JNK/JNK在藥物組,電針組和針刀組中有差異(P0.05),COX-2含量電針組具有差異(P0.05),在藥物組和針刀組中有顯著差異(P0.01);②與空白組比較,模型組大鼠黑質(zhì)內(nèi)TH表達(dá)顯著降低(P0.01)。與模型組比較,藥物組、電針組大鼠黑質(zhì)內(nèi)TH表達(dá)均升高(P0.05),針刀組大鼠黑質(zhì)內(nèi)TH表達(dá)顯著升高(P0.01);③與空白組比較,模型組大鼠黑質(zhì)內(nèi)Caspase-3表達(dá)升高(P0.05),Beclin-1表達(dá)顯著升高(P0.01),Bcl-2表達(dá)顯著下降(P0.01);與模型組比較,Caspase-3含量在藥物組和電針組中降低(P0.05),針刀組顯著降低(P0.01);Beclin-1含量在電針組表達(dá)降低(P0.05),在藥物組和針刀組中表達(dá)顯著降低(P0.01);Bcl-2在三組中表達(dá)均升高(P0.05)。(4)Real time-PCR檢測(cè)結(jié)果:與空白組比較,模型組大鼠黑質(zhì)內(nèi)DAT表達(dá)顯著降低(P0.01);與模型組比較,藥物組大鼠黑質(zhì)內(nèi)DAT表達(dá)顯著升高(P0.01),電針組和針刀組大鼠黑質(zhì)內(nèi)DAT表達(dá)均升高(P0.05)。(5)TUNEL染色:與空白組比較,模型組大鼠神經(jīng)元凋亡數(shù)顯著增加(P0.01);與模型組比較,藥物組、電針組及針刀針組大鼠神經(jīng)元凋亡數(shù)顯著降低(P0.01);結(jié)論:(1)針刀干預(yù)能有效減輕PD大鼠黑質(zhì)神經(jīng)元炎癥反應(yīng),在JNK通路上,其可能通過(guò)降低COX-2/p-c-Jun的活性,降低JNK磷酸化水平,提高TH的抗氧化活性而發(fā)揮對(duì)神經(jīng)元細(xì)胞保護(hù)作用。(2)針刀干預(yù)可使TH神經(jīng)元形態(tài)改善、數(shù)量增多以及TH陽(yáng)性細(xì)胞的表達(dá)增多,提高DAT的表達(dá),從而發(fā)揮對(duì)神經(jīng)元的保護(hù)作用。(3)針刀干預(yù)能有效的減輕PD大鼠黑質(zhì)神經(jīng)元的凋亡,其可能通過(guò)降低Caspase-3的促凋亡活性,激活Bcl-2的抗凋亡活性,抑制自噬基因Beclin-1活性,提高TH的抗氧化活性而發(fā)揮對(duì)神經(jīng)元細(xì)胞保護(hù)作用。(4)對(duì)JNK信號(hào)的通路的抑制可能是針刀發(fā)揮作用的機(jī)制之一。
[Abstract]:Objective: To observe the effect of needle knife intervention on the inflammation of substantia nigra, nerve cell apoptosis and related factors in Parkinson's disease model rats, and to explore the mechanism of JNK signaling pathway in the intervention of needle knife in Parkinson's disease. Methods: 72 male SD rats were divided into blank group, sham operation group, model group, drug group, electroacupuncture group and needle knife group. The PD model was established by injection of 6-OHDA, and the model was tested by apomorphine (APO). The blank group did not do any treatment. The injection method and location of the sham operation group and the model group were the same, only the same amount of normal saline containing 0.2% vitamin C was injected; after the work was made, the drug group was treated with 50mg/kg gavage, and the electroacupuncture group was carried out. Acupuncture treatment, needle knife group was given needle intervention. After four weeks of treatment, the test indexes included: (1) behavioral test: record the number of rotations in 60min in rats; (2) HE staining to observe the morphology of substantia nigra neurons; (3) double immunofluorescence double labeling method for the detection of substantia nigra COX-2/TH, p-c-Jun/TH expression; (4) Westernblot detection of substantia nigra JNK, P-JNK, p-c-Jun COX-2 and TH expression; (5) immunohistochemical detection of substantia nigra TH nerve cells; (6) Real-TimePCR detection of substantia nigra DATmRNA content; (7) TUNEL staining detection of apoptotic cells (8) immunofluorescence double labeling method for the detection of substantia nigra Caspase-3/TH (9) Westernblot detection of substantia nigra Caspase-3, Bcl-2, Beclin-1 expression: (1) behavioral test: model group appearance There was no difference in rotation circle before and after treatment (P0.05). The number of rotating rings in the drug group, the electroacupuncture group and the needle knife group were significantly reduced, the activity increased, and there were significant differences (P0.01) before and after intervention (P0.01). (2) morphological examination: (1) HE staining: the number of different intervention methods can cause the number of HE dyed black matter nerve cells. In the drug group, the number of substantia nigra neurons in the drug group was orderly, the number and the model group increased, the degree of cell degeneration was reduced, and the proliferation of glial cells was not obvious; the number of black matter neurons in the electroacupuncture group was more obvious than the model group, and the degree of cell degeneration was obviously reduced; the number and model of the black matter nerve cells in the needle knife group were the number and model. The type group was more obvious, the arrangement was more dense and the cell degeneration was reduced. (2) the immunohistochemical staining of the substantia nigra TH: the substantia nigra of the normal group and the sham operation group were all dense TH immunoreactive positive cells, brown and large, and the cell body was mainly round or oval, and the few were polygons. The model group had a small amount of substantia nigra and sparsely TH free The number of immunoreactive neurons in the drug group, the electroacupuncture group and the needle knife group increased more obviously, the body contour was clearly visible and the staining intensity increased. (3) the results of immunofluorescence double labelled melanin COX-2/TH, p-c-Jun/TH, Caspase-3/TH detection: the black of the normal group and the sham operation group. The cytoplasm of TH immunoreactive positive cells was green, the cell body was large, and the cell body was mainly round or oval, and COX-2, p-c-Jun, Caspase-3/TH were red fluorescence, and the expression was weak in quantity and intensity. The model group had a few and sparse TH immunoreactive neurons, green, swollen and denatured cells, or COX-2, p-c-Jun, Caspase-3, with red fluorescence, increased in quantity and intensity. The TH immunoreactive neurons in the drug group, the electroacupuncture group and the needle knife group were green, arranged in a more orderly manner, increased in number, reduced the degree of body degeneration, and increased in dyeing strength; meanwhile, COX-2, p-c-J UN, Caspase-3/TH, red fluorescence, compared with model group, decreased in quantity, and the intensity of dyeing decreased. Compared with the electroacupuncture group and the drug group, the number of TH neurons in the needle knife group was more and the degree of cell degeneration was lighter; the number of COX-2 and p-c-Jun in the needle knife group was less than the electroacupuncture group, but more than the drug group; the quantity of Caspase-3 in the drug group was more than the electroacupuncture group and the needle knife group. (3) Western blot detection results: (1) compared with the blank group, there was no significant difference in the content of JNK in the substantia nigra of the model group (P0.05); P-JNK, P-JNK/JNK, p-c-Jun expression increased (P0.05), and the COX-2 expression increased significantly (P0.01). Compared with the model group, there was no significant difference in the JNK content of the substantia nigra in the drug group, electroacupuncture group and the needle knife group (P0.05); compared with the model group, The contents of P-JNK and p-c-Jun were different in the drug group and the electroacupuncture group (P0.05), and there were significant differences in the needle knife group (P0.01). Compared with the model group, the P-JNK/JNK was different in the drug group, the electroacupuncture group and the needle knife group (P0.05), the COX-2 content in the electroacupuncture group was different (P0.05), and there was a significant difference between the drug group and the needle knife group (P0.01); (2) compared with the blank group, the model was compared with the blank group. The expression of TH in the substantia nigra of the rats was significantly decreased (P0.01). Compared with the model group, the expression of TH in the substantia nigra of the rats in the drug group and the electroacupuncture group increased (P0.05), and the TH expression in the substantia nigra of the Acupotomy rats increased significantly (P0.01). (3) the expression of Caspase-3 in the substantia nigra of the model group increased (P0.05), and the Beclin-1 expression increased significantly (P0.01), Bcl-2 expression was obvious. Decrease (P0.01). Compared with the model group, the content of Caspase-3 decreased (P0.05) in the drug group and the electroacupuncture group (P0.01), the expression of Beclin-1 decreased (P0.05) in the electroacupuncture group (P0.05), and the expression in the drug group and the needle knife group was significantly decreased (P0.01); Bcl-2 in the three groups increased (P0.05). (4) Real time-PCR detection results: and blank Compared with the model group, the expression of DAT in the substantia nigra was significantly decreased (P0.01). Compared with the model group, the expression of DAT in the substantia nigra of the drug group increased significantly (P0.01), and the expression of DAT in the substantia nigra of the electroacupuncture group and the Acupotomy group increased (P0.05). (5) TUNEL staining: compared with the blank group, the neuron apoptosis increased significantly (P0.01) in the model group (P0.01); compared with the model group, the rat model group was compared with the model group. Compared with the drug group, electroacupuncture group and needle knife group, the neuron apoptosis was significantly decreased (P0.01). Conclusion: (1) acupuncture intervention can effectively reduce the inflammatory response of the substantia nigra neurons in PD rats. On the JNK pathway, it may reduce the activity of COX-2/p-c-Jun, reduce the level of JNK phosphorylation and improve the antioxidant activity of TH to protect the neuron cells. (2) acupuncture intervention can improve the morphology of TH neurons, increase the number of TH positive cells, increase the expression of DAT, and improve the protective effect of the neurons. (3) acupuncture intervention can effectively reduce the apoptosis of the neurons in the substantia nigra of PD rats. It may pass the apoptotic activity of Caspase-3 and activate the anti apoptosis of Bcl-2. Activity, inhibit the activity of autophagy gene Beclin-1, improve the antioxidant activity of TH and play a protective role on neuron cell. (4) inhibition of JNK signaling pathway may be one of the mechanisms of acupotomy.
【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R245

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3 王偉;張光毅;;H_2O_2誘導(dǎo)培養(yǎng)的大鼠皮層神經(jīng)元凋亡與JNK信號(hào)通路的關(guān)系[A];中國(guó)生物化學(xué)與分子生物學(xué)會(huì)第八屆會(huì)員代表大會(huì)暨全國(guó)學(xué)術(shù)會(huì)議論文摘要集[C];2001年

4 范為民;王小琴;;JNK信號(hào)通路在慶大霉素誘導(dǎo)腎損傷大鼠細(xì)胞凋亡中的機(jī)制及左歸丸干預(yù)作用研究[A];第四屆全國(guó)中醫(yī)藥博士生優(yōu)秀論文專(zhuān)輯[C];2013年

5 劉燕;李強(qiáng);葛嬌;顧玲;郭霞;馬志貴;朱易萍;高舉;袁粒星;;茴香霉素通過(guò)活化p38-MAPK及JNK信號(hào)通路誘導(dǎo)糖皮質(zhì)激素耐藥的T-急性淋巴細(xì)胞白血病CEM-C1細(xì)胞生長(zhǎng)抑制及凋亡[A];中華醫(yī)學(xué)會(huì)第十七次全國(guó)兒科學(xué)術(shù)大會(huì)論文匯編（下冊(cè)）[C];2012年

6 杜仲燕;胡林峰;潘然;張福利;丁蕾;沃興德;竇曉兵;;基于JNK信號(hào)通路探究半胱氨酸在非酒精性脂肪性肝炎發(fā)病機(jī)制中的作用[A];第十二屆全國(guó)脂質(zhì)與脂蛋白學(xué)術(shù)會(huì)議論文匯編[C];2014年

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1 蘆娟;基于JNK信號(hào)通路探討針刀干預(yù)對(duì)帕金森病大鼠機(jī)制的研究[D];北京中醫(yī)藥大學(xué);2017年

2 羅明t ;桑葉有效部位降血糖作用與JNK信號(hào)通路的關(guān)系[D];廣州中醫(yī)藥大學(xué);2013年

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1 柴瀟瀟;紫杉醇誘導(dǎo)細(xì)胞凋亡過(guò)程中JNK信號(hào)通路的作用及NF-κBp65蛋白表達(dá)的變化[D];河北醫(yī)科大學(xué);2015年

2 牛志通;西格列汀對(duì)高糖誘導(dǎo)的大鼠腎小管上皮細(xì)胞JNK信號(hào)通路的影響[D];山西醫(yī)科大學(xué);2016年

3 陳冰;脂多糖協(xié)同多聚左旋精氨酸誘導(dǎo)NCI-H292細(xì)胞分泌IL-6、IL-8的JNK信號(hào)通路研究[D];安徽醫(yī)科大學(xué);2016年

4 李志敏;JNK信號(hào)通路參與雌二醇減輕皮瓣缺血再灌注損傷的研究[D];蘇州大學(xué);2016年

5 莊澤眾;跑臺(tái)訓(xùn)練對(duì)腦缺血損傷大鼠JNK信號(hào)通路和血管再生的影響[D];福建醫(yī)科大學(xué);2016年

6 楊浩;基于JNK信號(hào)通路探究溫陽(yáng)振衰顆粒對(duì)慢性心衰模型兔心肌細(xì)胞JNK表達(dá)及凋亡的影響[D];湖南中醫(yī)藥大學(xué);2015年

7 蘇曉明;三氧化二砷誘導(dǎo)雄激素非依賴(lài)前列腺癌PC-3細(xì)胞凋亡的JNK信號(hào)通路研究[D];大連醫(yī)科大學(xué);2013年

8 王昀;川芎嗪抗缺血再灌注損傷作用與JNK信號(hào)通路的相關(guān)性分析[D];廣州中醫(yī)藥大學(xué);2012年

9 傅應(yīng)亞;抑制JNK信號(hào)通路下調(diào)LRP的表達(dá)可增強(qiáng)順鉑化療敏感性[D];重慶醫(yī)科大學(xué);2014年

10 彭雷;JNK信號(hào)通路在亞砷酸鈉所致HELF細(xì)胞DNA斷裂損傷中的作用[D];南京醫(yī)科大學(xué);2008年

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