本文選題：脊髓損傷 + 雷帕霉素　； 參考：《吉林大學(xué)》2015年博士論文

【摘要】：脊髓損傷（spinal cord injury, SCI）的病理過(guò)程包括原發(fā)性和繼發(fā)性損傷，前者指暴力發(fā)生時(shí)脊髓的不可逆性損傷，后者是在此基礎(chǔ)上發(fā)生的局部缺血、水腫、電解質(zhì)紊亂等一系列生理生化反應(yīng)。缺血缺氧和炎癥反應(yīng)均能引起活性氧(reactive oxgen specieces，ROS)的產(chǎn)生和釋放，ROS大量堆積導(dǎo)致局部組織氧化損傷，神經(jīng)細(xì)胞發(fā)生凋亡和壞死。尋找有效藥物減少脊髓神經(jīng)元繼發(fā)性死亡，一直以來(lái)都是人們研究的熱點(diǎn)。 雷帕霉素（Rapamycin, Rap）是腎移植術(shù)后抗排斥反應(yīng)藥物，是一種高效的免疫抑制劑。近期發(fā)現(xiàn)，雷帕霉素又具有上調(diào)自噬的作用。中樞神經(jīng)系統(tǒng)創(chuàng)傷性疾病研究顯示，雷帕霉素會(huì)隨動(dòng)物種類和疾病模型不同而發(fā)揮截然相反的作用，脊髓損傷時(shí)，雷帕霉素是否具有神經(jīng)保護(hù)作用，目前尚無(wú)定論。 本研究建立大鼠脊髓損傷模型，觀察雷帕霉素對(duì)損傷脊髓是否具有保護(hù)作用；選擇H2O2氧化損傷PC12細(xì)胞模型，進(jìn)一步探討雷帕霉素對(duì)神經(jīng)細(xì)胞的保護(hù)作用機(jī)制，通過(guò)NF-kB和NLRP3炎癥小體表達(dá)情況，初步探索自噬和炎癥之間相互作用的關(guān)系，論文分以下兩部分闡述。 第一部分雷帕霉素對(duì)脊髓撞擊損傷大鼠的神經(jīng)保護(hù)作用 目的：建立大鼠脊髓損傷模型，觀察雷帕霉素是否具有上調(diào)自噬、下調(diào)炎癥以及抑制凋亡的神經(jīng)保護(hù)作用。 方法：Wistar雌性大鼠45只。開(kāi)椎板假手術(shù)組3只，42只大鼠制備脊髓撞擊損傷模型，隨機(jī)分成雷帕霉素治療組和損傷對(duì)照組。術(shù)后3d、7d、14d取材，進(jìn)行H-E染色、ED-1染色、GFAP免疫熒光染色，觀察脊髓損傷病理學(xué)變化；酶組織化學(xué)法檢測(cè)組織髓過(guò)氧化物酶(MPO)濃度；Western blot法檢測(cè)炎性因子TNF-α、IL-1β、自噬相關(guān)蛋白Beclin-1的表達(dá)；TUNEL法檢測(cè)脊髓組織細(xì)胞凋亡指數(shù)；NF免疫組化染色觀察脊髓組織中存活神經(jīng)元。 結(jié)果：成功建立大鼠脊髓撞擊損傷模型，術(shù)后3d雷帕霉素治療組MPO值 低于損傷組(P0.05)，Western blot結(jié)果顯示雷帕霉素組炎性因子IL-lβ，TNF-α 表達(dá)低于損傷組(P0.05)，自噬相關(guān)蛋白Beclin-1表達(dá)高于損傷組。術(shù)后7d ED-1陽(yáng)性小膠質(zhì)細(xì)胞在雷帕霉素組明顯少于損傷對(duì)照組(P0.05)，術(shù)后14d GFAP熒 光染色陽(yáng)性星型膠質(zhì)細(xì)胞，雷帕霉素組有減少趨勢(shì)，但與損傷組之間無(wú)顯著性差異。H-E染色結(jié)果顯示，在損傷區(qū)與正常脊髓交界處，損傷組炎癥細(xì)胞數(shù)目較多。TUNEL結(jié)果顯示，雷帕霉素組凋亡細(xì)胞數(shù)目明顯減少(P0.05)。NF染色顯示，雷帕霉素組神經(jīng)元數(shù)目明顯多于單純損傷組(P0.05)。 結(jié)論：雷帕霉素作用于大鼠脊髓撞擊損傷，不僅可以上調(diào)自噬；而且能夠抑制小膠質(zhì)細(xì)胞活化和增殖、減少中性粒細(xì)胞及淋巴細(xì)胞浸潤(rùn)，減少炎性因子表達(dá)，減輕了神經(jīng)組織繼發(fā)性炎癥反應(yīng)；雷帕霉素最終減少凋亡細(xì)胞數(shù)量，增加神經(jīng)元存活，對(duì)損傷脊髓具有保護(hù)作用。 第二部分雷帕霉素對(duì)氧化應(yīng)激損傷PC12細(xì)胞的保護(hù)作用及機(jī)制研究 目的：觀察雷帕霉素預(yù)處理對(duì)氧化應(yīng)激損傷PC12細(xì)胞的保護(hù)作用，從自噬和炎癥之間的關(guān)聯(lián)信號(hào)通路探討雷帕霉素保護(hù)作用的機(jī)制。 方法：實(shí)驗(yàn)共分五組：正常PC12細(xì)胞組；H2O2+PC12組；RAP+H2O2+PC12組；RAP+SN50+H2O2+PC12組；3-MA+H2O2+PC12組，制備H2O2氧化損傷PC12細(xì)胞模型。MTT法檢測(cè)各組細(xì)胞存活率；光學(xué)顯微鏡觀察細(xì)胞形態(tài)學(xué)變化；MDC染色觀察細(xì)胞自噬囊泡變化；ROS染色觀察細(xì)胞活性氧變化；ELISA法檢測(cè)培養(yǎng)上清和細(xì)胞內(nèi)炎性因子TNF-α、IL-1β變化；Western blot法檢測(cè)自噬相關(guān)蛋白Beclin-1、LC3，促凋亡蛋白Bax、Cleave-caspase3，炎癥信號(hào)通路NF-kB、炎癥小體NLRP3、炎性因子IL-1β表達(dá)變化。 結(jié)果：200μM H2O2處理PC12細(xì)胞24h，成功構(gòu)建氧化損傷細(xì)胞模型。MTT法結(jié)果提示Rap和Rap+SN50組與H2O2組比較，細(xì)胞存活率增加；3-MA組與H2O2組比較，細(xì)胞存活率減少。ROS染色顯示H2O2組和3-MA組的平均熒光 強(qiáng)度與正常組比較明顯增高，Rap和Rap+SN50組與H2O2組比較熒光強(qiáng)度明顯減少。MDC染色后發(fā)現(xiàn)H2O2組自噬囊泡數(shù)量較多；Rap和Rap+SN50組細(xì)胞內(nèi)自噬囊泡熒光顆粒明顯增強(qiáng)；3-MA組細(xì)胞自噬囊泡數(shù)量較少。ELISA結(jié)果顯示H2O2組細(xì)胞損傷后培養(yǎng)上清和細(xì)胞內(nèi)TNF-α、IL-1β分泌水平明顯增高；而Rap和Rap+SN50組TNF-α、IL-1β分泌水平降低。Western blot結(jié)果顯示Rap和Rap+SN50組自噬相關(guān)蛋白LC3和Beclin-1的表達(dá)明顯高于H2O2組，而3-MA組LC3和Beclin-1的表達(dá)明顯低于H2O2組；炎癥通路蛋白NF-кB和NLRP3表達(dá)在Rap和Rap+SN50組明顯低于H2O2組，而3-MA組NF-кB的表達(dá)明顯高于H2O2組；促凋亡蛋白Bax和Cleave-caspase3表達(dá)在Rap和Rap+SN50組低于H2O2組，而3-MA組Bax和Cleave-caspase3的表達(dá)則明顯高于H2O2組。結(jié)論：在H2O2氧化損傷PC12細(xì)胞模型中，雷帕霉素通過(guò)上調(diào)自噬相關(guān)蛋白LC3和Beclin-1表達(dá)，減少損傷線粒體ROS釋放，下調(diào)NF-kB信號(hào)通路和NLRP3炎癥小體活化，，減少炎性因子TNF-α、IL-1β表達(dá)，雷帕霉素下調(diào)促凋亡蛋白Bax和Cleave-caspase3表達(dá)，增加PC12細(xì)胞存活，具有神經(jīng)細(xì)胞保護(hù)作用。 綜上所述，我們?cè)诖笫篌w內(nèi)和體外培養(yǎng)PC12細(xì)胞實(shí)驗(yàn)證實(shí)，雷帕霉素具有神經(jīng)保護(hù)作用。初步探討其可能存在的機(jī)制：脊髓損傷后，雷帕霉素上調(diào)自噬水平，減少ROS產(chǎn)生，通過(guò)NF-kB和NLRP3信號(hào)通路減少炎性因子的產(chǎn)生和釋放，雷帕霉素下調(diào)促凋亡信號(hào)，減少死亡細(xì)胞數(shù)量；在脊髓損傷區(qū)域，由于死亡細(xì)胞炎性因子釋放減少，下調(diào)了周邊炎性細(xì)胞活化和增殖遷移，減小局部炎癥反應(yīng)強(qiáng)度，雷帕霉素減少凋亡細(xì)胞數(shù)量，保護(hù)了殘存神經(jīng)細(xì)胞。
[Abstract]:The pathological process of spinal cord injury (SCI) includes primary and secondary injuries. The former refers to the irreversible injury of the spinal cord at the time of violence. The latter is a series of physiological and biochemical reactions, such as local ischemia, edema, and electrolyte disturbance on this basis. The oxygen deficiency and the inflammatory response can cause the reactive oxygen species (reactive oxge). The production and release of n Specieces, ROS, a large accumulation of ROS causes oxidative damage in local tissues and apoptosis and necrosis of nerve cells. Finding effective drugs to reduce secondary death of spinal neurons has always been a hot spot of research.
Rapamycin (Rap) is an anti rejection drug after renal transplantation and is an effective immunosuppressant. Recently, rapamycin has the effect of increasing autophagy. The central nervous system traumatic disease study shows that rapamycin will play a very opposite effect on the animal species and disease model, spinal cord injury. Whether rapamycin has neuroprotective effects is uncertain.
In this study, the rat spinal cord injury model was established to observe the protective effect of rapamycin on the injured spinal cord, and to select the H2O2 oxidative damage PC12 cell model to further explore the protective mechanism of rapamycin on the nerve cells, and to explore the interaction between autophagy and inflammation through the expression of NF-kB and NLRP3 corpuscles. The thesis is divided into two parts.
Part 1 the neuroprotective effect of rapamycin on spinal cord impact injury in rats
Objective: to establish a rat spinal cord injury model and observe whether rapamycin has the neuroprotective effect of upregulated autophagy, down-regulation of inflammation and inhibition of apoptosis.
Methods: 45 Wistar female rats. 3 rats in the sham operation group and 42 rats were prepared for the spinal cord impact injury model, which were randomly divided into rapamycin treatment group and injury control group. After operation, 3D, 7d, 14d were obtained, H-E staining, ED-1 staining, and GFAP immunofluorescence staining were used to observe the pathological changes of spinal cord injury; enzyme histochemical method was used to detect tissue medullary tissue. The concentration of oxide enzyme (MPO) and Western blot method were used to detect the expression of inflammatory factor TNF- alpha, IL-1 beta, autophagy related protein Beclin-1; TUNEL method was used to detect the apoptosis index of spinal cord tissue; NF immunohistochemical staining was used to observe the survival neurons in the spinal cord tissue.
Results: the spinal cord impact injury model was successfully established, and the MPO value of rapamycin treated group 3D after operation.
Lower than the injury group (P0.05), Western blot results showed rapamycin inflammatory factor IL-l beta, TNF- alpha
The expression of autophagy related protein Beclin-1 was higher than that of injury group (P0.05), and the 7d ED-1 positive microglia in rapamycin group after operation was significantly less than that of the injury control group (P0.05), and 14d GFAP fluorescence after operation.
In the light staining positive astrocytes, there was a decreasing trend in the rapamycin group, but there was no significant difference between the injured group and the injured group. The results of.H-E staining showed that the number of inflammatory cells in the injured group and normal spinal cord was more.TUNEL. The number of apoptotic cells in the rapamycin group decreased significantly (P0.05).NF staining, and the rapamycin group The number of neurons was significantly more than that of the simple injury group (P0.05).
Conclusion: rapamycin can not only increase autophagy, but also inhibit the activation and proliferation of microglia, reduce the infiltration of neutrophils and lymphocytes, reduce the expression of inflammatory factors and reduce the secondary inflammatory response in the nerve tissue, and reparamycin can eventually reduce the number of apoptotic cells and increase the nerve. Yuan Cunhuo has a protective effect on the injury of the spinal cord.
The second part is the protective effect and mechanism of rapamycin on PC12 cells injured by oxidative stress.
Objective: To observe the protective effect of rapamycin preconditioning on PC12 cells damaged by oxidative stress, and to explore the mechanism of the protective effect of rapamycin from the correlation signal pathway between autophagy and inflammation.
Methods: the experiment was divided into five groups: normal PC12 cell group, group H2O2+PC12, group RAP+H2O2+PC12, group RAP+SN50+H2O2+PC12, group RAP+SN50+H2O2+PC12; 3-MA+H2O2+PC12 group, H2O2 oxidative damage PC12 cell model.MTT method was used to detect the cell survival rate of each group; optical microscope observation of cell morphological changes; MDC staining observation of cell autophagic vesicles; ROS staining The changes of active oxygen in cells were observed. ELISA assay was used to detect TNF- alpha and IL-1 beta in cultured supernatant and intracellular inflammatory factors, and Western blot was used to detect autophagy related protein Beclin-1, LC3, apoptotic protein Bax, Cleave-caspase3, NF-kB of inflammatory signaling pathway, NLRP3 inflammatory body, and changes in the expression of inflammatory cell IL-1 beta.
Results: the PC12 cell 24h was treated with 200 M H2O2, and the.MTT method of the oxidative damage cell model was successfully constructed. The cell survival rate of Rap and Rap+SN50 groups was increased compared with that of H2O2 group, and the 3-MA group was compared with the H2O2 group, and the cell survival rate reduced by.ROS staining showed the average fluorescence of the H2O2 group and the group.
The intensity was significantly higher than that in the normal group. The fluorescence intensity of Rap and Rap+SN50 groups was significantly lower than that in the H2O2 group. The number of autophagic vesicles in the H2O2 group was more than that in the H2O2 group. The intracellular autophagic vesicles in the Rap and Rap+SN50 groups were obviously enhanced, and the less.ELISA results of the autophagic vesicles in the 3-MA group showed that the cells in the H2O2 group were damaged after the cell injury. The secretion of TNF- alpha and IL-1 beta in both the clear and the cells increased significantly, while the secretion level of TNF- alpha and IL-1 beta in the Rap and Rap+SN50 groups decreased by.Western blot and showed that the expression of LC3 and Beclin-1 were significantly higher in Rap and Rap+SN50 groups than those in the group. The expression of NF- and B in group 3-MA was significantly higher than that in group H2O2, and the expression of Bax and Cleave-caspase3 in 3-MA group was significantly higher than that in group H2O2, and the expression of Bax and Cleave-caspase3 in Rap and Rap+SN50 groups was lower than that of the H2O2 group, while the expression of NF- and B was significantly higher than that of the group of H2O2. The expression of autophagy related protein LC3 and Beclin-1 reduces the release of ROS in damaged mitochondria, reduces the activation of NF-kB signaling pathway and NLRP3 inflammatory corpuscle, reduces the expression of inflammatory factor TNF- alpha and IL-1 beta, reduces the expression of Bax and Cleave-caspase3, and increases the survival of PC12 cells, and has the protective effect of nerve cells.
To sum up, our experiment in rat and in vitro culture of PC12 cells confirmed that rapamycin has a neuroprotective effect. Preliminary study of its possible mechanism: after spinal cord injury, rapamycin up-regulated the autophagy level, reduced ROS production, reduced the production and release of inflammatory factors through the NF-kB and NLRP3 signaling pathways, and down regulation of rapamycin The apoptotic signal reduces the number of dead cells; in the region of spinal cord injury, the reduction of inflammatory factors in the dead cells reduces the activation and proliferation of peripheral inflammatory cells, reduces the intensity of local inflammatory reaction, reduces the number of apoptotic cells by rapamycin, and protects the remnants of the deity cells.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R651.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Hai-Hu Hao;Li Wang;Zhi-Jian Guo;Lang Bai;Rui-Ping Zhang;Wei-Bing Shuang;Yi-Jia Jia;Jie Wang;Xiao-Yu Li;Qiang Liu;;Valproic acid reduces autophagy and promotes functional recovery after spinal cord injury in rats[J];Neuroscience Bulletin;2013年04期

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