本文選題：脊髓損傷 + 蕓香苷��； 參考：《山西醫(yī)科大學》2017年博士論文

【摘要】：目的:在改良ALLEN法建立的大鼠脊髓損傷動物模型上,檢測蕓香苷是否能促進脊髓損傷后大鼠的神經(jīng)功能恢復,改善脊髓損傷后炎癥因子浸潤,減輕炎癥反應,并檢測蕓香苷對脊髓損傷后PI3K/AKT信號通路的影響,來探討蕓香苷發(fā)揮脊髓損傷后神經(jīng)保護作用的作用機制。方法:1.動物模型和分組:120只雄性成年SD大鼠,隨機分成4組,每組30只。假手術組(CON組):切除椎板,不損傷脊髓,注射1ml的DMSO;脊髓損傷組(SCI組):根據(jù)ALLEN法建立脊髓損傷大鼠模型(T10節(jié)段),注射1ml的DMSO;甲強龍治療組(MP組):脊髓損傷同SCI組,腹腔內(nèi)注射甲強龍30mg/kg;蕓香苷治療組(RT100組):脊髓損傷SCI組,腹腔內(nèi)注射蕓香苷100mg/kg。2.每組10只大鼠,在脊髓損傷后的第1、2、3周,采用體感誘發(fā)電位對大鼠脊髓功能進行客觀檢測,觀察指標選用潛伏期和波幅。在脊髓損傷后的第1、7、14、21、28天,采用BBB評分、斜坡實驗和改良Tarlov評分等行為學方法評價大鼠后肢運動功能和身體平衡能力。3.取材:在大鼠脊髓損傷后24小時處死動物,每組5只大鼠取新鮮脊髓用于含水量檢測;每組5只大鼠取新鮮脊髓用于ELISA、明膠酶譜法和光譜分析法;每組5只大鼠取新鮮脊髓用Western Blot實驗;每組5只大鼠經(jīng)心臟灌注4%多聚甲醛內(nèi)固定后取脊髓,放在相同固定液再固定24小時后,石蠟包埋切片用于免疫組織化學染色和HE染色。4.采用免疫組化染色法測定脊髓組織中IL-1β、TNF-α、MCP-1、IL-10、TGF-β、GFAP、IBA-1、Bax、Bcl-2的表達,采用HE染色觀察脊髓組織形態(tài)和病理表現(xiàn)。5.采用Hsu公式測定脊髓損傷后脊髓組織含水量,采用酶聯(lián)免疫吸附法(ELISA)測定脊髓組織中MIP-2的含量,明膠酶譜法測定脊髓組織中MMP-9的含量,光譜分析法測定IKKβ激酶活性。6.采用Western blot法檢測脊髓組織中的P-AKT、NF-κB P65、Bax、Bcl-2的表達。結(jié)果:1.體感誘發(fā)電位結(jié)果:SCI組在第1、2、3周的SEP潛伏期與CON組比較明顯延長,SEP的波幅均明顯下降;與SCI組相比,MP組和RT100組在第1、2、3周潛伏期均呈現(xiàn)不同程度的縮短,波幅均呈現(xiàn)不同程度的升高,差異有統(tǒng)計學意義(p0.05)。2.BBB、斜坡實驗、改良Tarlov功能評分的結(jié)果:在脊髓損傷后的每個檢測時間點,SCI組大鼠的評分均明顯低于CON組;與SCI組相比,MP組和RT100組大鼠的評分雖然在第1和第7天無明顯差異,但在第14、21、28天均明顯提高,差異有統(tǒng)計學意義(p0.05)。3.脊髓含水量的結(jié)果:與CON組相比,SCI組的含水量明顯升高,差異有統(tǒng)計學意義(p0.05);與SCI組相比,RT100組脊髓含水量明顯降低,差異有統(tǒng)計學意義(p0.01);MP組和RT100組相比,治療效果相當,在脊髓組織含水量上沒有差異,無統(tǒng)計學意義(p0.05)。4.HE染色結(jié)果:與CON組相比,SCI組中的神經(jīng)元細胞數(shù)量明顯減少,差異有統(tǒng)計學意義(p0.05);與SCI組相比,MP組和RT100組神經(jīng)元細胞數(shù)目減少受到抑制,差異有統(tǒng)計學意義(p0.05);MP組和RT100組相比,在抑制神經(jīng)元數(shù)目減少方面,效果相當,沒有統(tǒng)計學意義(p0.05)。5.免疫組化染色炎癥因子表達的結(jié)果:與CON組相比,SCI組中的IL-1β、TNF-α、MCP-1水平顯著升高,差異有統(tǒng)計學意義(p0.05);與SCI組相比,RT100組和MP組IL-1β、TNF-α、MCP-1表達水平明顯降低,差異有統(tǒng)計學意義(p0.05);與CON組相比,SCI組中的IL-10、TGF-β水平明顯升高,差異有統(tǒng)計學意義(p0.05);與SCI組相比,RT100組和MP組中IL-10、TGF-β表達水平明顯增高,差異有統(tǒng)計學意義(p0.05)。6.ELISA檢測MIP-2含量的結(jié)果:與CON組相比,SCI組中MIP-2的水平明顯增強,差異有統(tǒng)計學意義(p0.05);與SCI組相比,MP組和RT100組中MIP-2水平明顯降低,差異有統(tǒng)計學意義(p0.05,p0.01);MP組和RT100組中MIP-2表達水平?jīng)]有明顯差異(p0.05)。7.明膠酶譜法測定MMP-9活性的結(jié)果:SCI組和CON組相比,MMP-9的活性和生成量都明顯升高,差異有統(tǒng)計學意義(p0.05);與SCI組相比,MP組和RT100組中MMP-9的生成量和活性明顯降低,差異有統(tǒng)計學意義(p0.01);MP組和RT100組相比,脊髓組織中MMP-9的活性和生成量沒有差異,無統(tǒng)計學意義(p0.05)。8.免疫組化染色檢測GFAP和IBA-1表達的結(jié)果:SCI組與CON組相比,GFAP、IBA-1表達水平明顯增高,差異有統(tǒng)計學意義(p0.05);與SCI組相比,RT100組和MP組GFAP和IBA-1表達降低,差異有統(tǒng)計學意義(p0.05)。9.光譜分析檢測IKKβ激酶活性結(jié)果:在CON組中,脊髓組織的IKKβ激酶活性較低,SCI組相比CON組中IKKβ激酶活性明顯增高,差異有統(tǒng)計學意義(p0.05);MP組和RT100組IKKβ激酶活性相比SCI組明顯降低,差異有統(tǒng)計學意義(P0.05);MP組和RT100組相比在抑制IKKβ激酶活性上差異無統(tǒng)計學意義(P0.05)。10.免疫組化染色和Western blot檢測Bax和Bcl-2表達的結(jié)果:SCI組與CON組相比,Bax表達水平明顯增高,而Bcl-2表達明顯降低,差異有統(tǒng)計學意義(p0.05);與SCI組相比,RT100組和MP組Bax的表達降低,Bcl-2的表達升高,差異有統(tǒng)計學意義(p0.05);RT100組和MP組相比,差異沒有統(tǒng)計學意義(p0.05)。11.Western blot p-Akt和NF-κB P65表達的結(jié)果:CON組中p-Akt、NF-κB P65蛋白的表達較少,SCI組和CON組相比,p-Akt、NF-κB P65蛋白表達明顯增高,差異有統(tǒng)計學意義(p0.05);與SCI組相比,藥物治療組在p-Akt、NF-κB P65蛋白的生成量上都有不同程度的抑制作用,RT100組和MP組能夠調(diào)節(jié)p-Akt、NF-κB P65蛋白的表達,差異有統(tǒng)計學意義(p0.05);MP組和RT100組在表達p-Akt、NF-κB P65蛋白方面無顯著差異,沒有統(tǒng)計學意義(p0.05)。結(jié)論:1.蕓香苷能夠縮短脊髓損傷大鼠SEP潛伏期,增加波幅,改善脊髓損傷后大鼠的后肢運動能力和身體平衡能力,對脊髓損傷后大鼠的神經(jīng)功能有保護作用。2.蕓香苷能夠減輕脊髓損傷后的組織水腫,減少神經(jīng)元細胞的損傷,抑制促炎因子IL-1β和TNF-α的表達,減少趨化因子MCP-1、MIP-2和MMP-9的產(chǎn)生,促進抗炎因子IL-10和TGF-β因子的表達,抑制星形膠質(zhì)細胞和小膠質(zhì)細胞的激活,減輕炎癥反應。3.蕓香苷能夠抑制脊髓損傷后PI3K/AKT信號通路,降低IKKβ激酶活性,減少p-Akt和NF-κB P65蛋白的表達,下調(diào)Bax的表達,上調(diào)Bcl-2的表達,降低Bax/Bcl-2的比例,抑制細胞凋亡。蕓香苷可能是通過PI3K/AKT實現(xiàn)對脊髓損傷大鼠的神經(jīng)保護作用。
[Abstract]:Objective: to determine whether rutin can promote the recovery of nerve function in rats after spinal cord injury, improve the infiltration of inflammatory factors after spinal cord injury, reduce the inflammatory response, and detect the effect of rutin on the PI3K/AKT signaling pathway after spinal cord injury to explore the effect of rutin on the spinal cord injury in the rat model of spinal cord injury established by the improved ALLEN method. Methods: 1. animal models and groups: 1. animal models and groups: 120 adult male adult SD rats were randomly divided into 4 groups, 30 in each group. The sham operation group (group CON): the laminectomy, the spinal cord injury, the injection of 1ml DMSO, the spinal cord injury group (group SCI): the ALLEN method was used to establish the rat model of spinal cord injury (T10 segment), the DMSO of 1ml and a strength of 1ml Dragon treatment group (group MP): spinal cord injury and SCI group, intraperitoneal injection of methylprednisolone 30mg/kg, rutin treatment group (group RT100): spinal cord injury SCI group, intraperitoneal injection of rutin 100mg/kg.2. in each group of 10 rats, after spinal cord injury week 1,2,3, using somatosensory evoked potential to detect the function of spinal cord in rats, observe the incubation period of the incubation period And amplitude. On day 1,7,14,21,28 after spinal cord injury, the BBB score, slope experiment and improved Tarlov score were used to evaluate the motor function and body balance of the rats. The animals were killed 24 hours after the spinal cord injury in rats. 5 rats in each group were used for the detection of water content, and 5 rats in each group were taken out. The fresh spinal cord was used for ELISA, gelatin Zymogram and spectral analysis. 5 rats in each group were taken Western Blot experiment with fresh spinal cord. 5 rats in each group were fixed in the same fixed solution for 24 hours after perfusion of 4% polyformaldehyde, and the paraffin embedded sections were used for immunohistochemical staining and HE staining for.4. immunization. The expression of IL-1 beta, TNF- a, MCP-1, IL-10, TGF- beta, GFAP, IBA-1, Bax, Bcl-2 in spinal cord tissue was determined by staining, and HE staining was used to observe the morphological and pathological manifestations of spinal cord tissue and pathology. The content of spinal cord tissue was measured by the Hsu formula. The content of spinal cord tissue was determined by enzyme linked immunosorbent assay, and gelatinase assay was used to determine the content of spinal cord tissue. The content of MMP-9 in spinal cord tissue, the determination of the activity of IKK beta kinase activity by spectral analysis and Western blot method to detect P-AKT in spinal cord tissue, NF- kappa B P65, Bax, Bcl-2 expression. Results: 1. somatosensory evoked potential results: the incubation period in the SCI group was significantly longer than that of the group, and the amplitude of NF- kappa was obviously decreased; compared with the group The latency of the RT100 group in the 1,2,3 week showed a different degree of shortening, and the amplitude of the wave increased in varying degrees. The difference was statistically significant (P0.05).2.BBB, the slope experiment, and the result of improving the Tarlov function score: the score of the SCI group was significantly lower than that of the CON group at each detection time point after spinal cord injury, and the MP group and RT were compared with the SCI group. There was no significant difference between the 100 groups in the first and seventh days, but the difference was significantly higher in day 14,21,28. The difference was statistically significant (P0.05) the result of the water content in the.3. spinal cord. Compared with the CON group, the water content of the SCI group was significantly higher, the difference was statistically significant (P0.05). Compared with the SCI group, the water content of the RT100 group was significantly lower than that in the RT100 group, and the difference was unified. Study significance (P0.01); MP group and RT100 group compared with the treatment effect, there was no difference in the water content of the spinal cord, no statistical significance (P0.05).4.HE staining results: compared with the CON group, the number of neurons in the SCI group decreased significantly (P0.05), and the number of neurons in MP and RT100 groups decreased compared with the SCI group. The difference was statistically significant (P0.05), and in group MP and group RT100, the effect was similar in reducing the number of neurons, and there was no statistically significant (P0.05).5. immunohistochemical staining of the expression of inflammatory factors: compared with the CON group, the level of IL-1 beta, TNF- alpha, MCP-1 in the SCI group was significantly higher, and the difference was statistically significant (P0.05); and SCI with SCI. Compared with group RT100 and group MP, the expression level of IL-1 beta, TNF- alpha and MCP-1 was significantly lower, and the difference was statistically significant (P0.05). Compared with the CON group, the IL-10 and TGF- beta levels in SCI group were significantly higher, and the difference was statistically significant (P0.05). The results of ELISA detection of MIP-2 content: compared with the CON group, the level of MIP-2 in the SCI group was obviously enhanced, and the difference was statistically significant (P0.05). Compared with the SCI group, the MIP-2 level of MP and RT100 groups was significantly decreased, the difference was statistically significant (P0.05, P0.01). The results of MP-9 activity: the activity and production of MMP-9 increased significantly in the group SCI and the CON group, and the difference was statistically significant (P0.05). Compared with the SCI group, the amount and activity of MMP-9 in the MP group and RT100 group decreased significantly (P0.01), and there was no difference in the activity and production of the spinal cord between the MP group and the RT100 group. There was no statistical significance (P0.05).8. immunohistochemical staining to detect the expression of GFAP and IBA-1: the expression level of GFAP, IBA-1 in SCI group was significantly higher than that in CON group, and the difference was statistically significant (P0.05). The difference was statistically significant compared with the SCI group. Results: in the group CON, the activity of IKK beta kinase in the spinal cord was lower, and the activity of IKK beta kinase in the group SCI was significantly higher than that in the group CON (P0.05), and the IKK beta kinase activity in the MP group and RT100 group was significantly lower than that in the SCI group, and the difference was statistically significant (P0.05), and there was no statistical difference between the MP group and the group in the MP group. P0.05.10. immunohistochemical staining and Western blot were used to detect the expression of Bax and Bcl-2: the Bax expression level was significantly higher in the SCI group than that in the CON group, but the Bcl-2 expression was significantly lower, and the difference was statistically significant (P0.05). Compared with the MP group, the difference was not statistically significant (P0.05).11.Western blot p-Akt and NF- kappa B P65 expression results: CON group p-Akt, NF- kappa B protein expression was less. RT100 and MP groups could regulate the expression of p-Akt, NF- kappa B P65 protein, and the difference was statistically significant (P0.05). There was no significant difference in the expression of p-Akt, NF- kappa B protein in MP and RT100 groups. Conclusion: 1. rutin can shorten the potential of spinal cord injury rats. .2. rutin can reduce the tissue edema after spinal cord injury, reduce the damage of neuron cells, inhibit the expression of IL-1 beta and TNF- alpha, and reduce the chemokine MCP-1, MI, and reduce the chemokine MCP-1, MI The production of P-2 and MMP-9 promotes the expression of anti-inflammatory factors IL-10 and TGF- beta factors, inhibits the activation of astrocytes and microglia, and reduces the inflammatory reaction.3. rutin can inhibit the PI3K/AKT signaling pathway after spinal cord injury, reduces the activity of IKK beta kinase, reduces the expression of p-Akt and NF- kappa B P65 protein, down regulate the expression of Bax, and up regulation of the expression of Bax. Rutin could reduce the proportion of Bax/Bcl-2 and inhibit cell apoptosis. Rutin may be a neuroprotective effect of PI3K/AKT on spinal cord injury in rats.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R651.2

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8 鄭穎t，

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