本文選題：青藤堿衍生物4EDB + 青藤堿��； 參考：《江蘇大學(xué)》2017年碩士論文

【摘要】：目的:以青藤堿(sinomenine,SIN)為原料,合成一種新的青藤堿衍生物—4EDB,并觀察青藤堿衍生物4EDB對(duì)實(shí)驗(yàn)性自身免疫性心肌炎(experimental autoimmune myocarditis,EAM)的治療效果,同時(shí)對(duì)4EDB抗炎機(jī)制進(jìn)行初步研究。方法:1.青藤堿衍生物4EDB的合成、鑒定以及抗炎活性檢測:對(duì)青藤堿A環(huán)4號(hào)位置進(jìn)行修飾,合成青藤堿衍生物4EDB,并通過氫譜和質(zhì)譜進(jìn)行鑒定,利用高效液相色譜法(hi gh performance liquid chromatography,HPLC)檢測4EDB的純度。建立膿毒血癥小鼠模型,給予不同濃度青藤堿和4EDB進(jìn)行灌胃治療,觀察小鼠的生存率,初步評(píng)價(jià)抗炎效果。2.4EDB對(duì)EAM的治療作用:建立EAM模型小鼠,再次免疫以后用4EDB進(jìn)行小鼠灌胃治療并觀察小鼠狀態(tài)。21天后處死小鼠,取出心肌組織,進(jìn)行HE染色與天狼腥紅染色以及提取RNA。通過HE染色獲取病理評(píng)分,天狼腥紅染色評(píng)價(jià)纖維化,實(shí)時(shí)定量熒光PCR(quantitative real-time PCR,qRT-PCR)檢測心肌組織中的炎癥因子IL-lβ、IL-6、IL-17A和TGF-β表達(dá)水平。取小鼠眼球血,離心收集上清,利用酶聯(lián)免疫吸附試驗(yàn)(enzymes linked immunosorbent assay,ELISA)檢測小鼠血清中炎癥因子IL-lβ、IL-6、IL-17A、IL-4和IFN-γ的表達(dá)水平。3.4EDB治療EAM的機(jī)制探討:以RAW264.7細(xì)胞為研究對(duì)象,利用流式細(xì)胞儀(flow cytometry,FCM)檢測4EDB對(duì)巨噬細(xì)胞凋亡的影響;利用Transwell實(shí)驗(yàn)檢測4EDB對(duì)巨噬細(xì)胞趨化的影響。結(jié)果:1.成功制備青藤堿衍生物4EDB,純度達(dá)到99.59%;通過膿毒血癥模型實(shí)驗(yàn)發(fā)現(xiàn)4EDB處理組小鼠的存活率高于青藤堿組,并且5 mg/kg的4EDB處理組抗炎效果較優(yōu);2.4EDB治療EAM模型小鼠:通過心肌組織病理評(píng)分,我們發(fā)現(xiàn)5 mg/kg的4EDB能夠緩解EAM的進(jìn)程。ELISA和qRT-PCR實(shí)驗(yàn)結(jié)果顯示4EDB可以明顯減少EAM小鼠血清中炎癥因子IL-17A、IL-lβ、IL-6的含量,而且天狼腥紅染色結(jié)果顯示4EDB能夠緩解EAM小鼠的心肌纖維化程度;3.流式細(xì)胞儀檢測和Transwell結(jié)果顯示10 nmol/mL及其以下濃度的4EDB不僅能夠誘導(dǎo)RAW264.7細(xì)胞的凋亡,也能夠抑制RAW264.7細(xì)胞的遷移。結(jié)論:1.合成一種新的青藤堿衍生物4EDB,且純度達(dá)到99.59%;與青藤堿相比,4EDB具有較好的抗炎效果;2.4EDB能夠下調(diào)炎癥因子IL-lβ、IL-6、IL-17A的水平,緩解心肌纖維化進(jìn)展,從而緩解EAM小鼠心肌組織的損傷;3.4EDB對(duì)EAM的治療作用可能是通過誘導(dǎo)心肌組織巨噬細(xì)胞凋亡以及抑制單核/巨噬細(xì)胞遷移發(fā)揮免疫抑制作用。
[Abstract]:Aim: to synthesize a new sinomenine derivative -4EDBs from sinomenine sinensis, and to observe the therapeutic effect of sinomenine derivative 4EDB on experimental autoimmune myocarditis, and to study the mechanism of 4EDB anti-inflammation.Method 1: 1.Synthesis, Identification and Anti-inflammatory activity Detection of Sinomenine Derivatives 4EDB: sinomenine Derivatives 4EDBs were synthesized by modifying the position of Sinomenine A Ring 4, and identified by hydrogen spectrum and mass spectrometry.High performance liquid chromatography (HPLC) was used to determine the purity of 4EDB.The sepsis mice model was established and the mice were treated with different concentrations of sinomenine and 4EDB by gavage. The survival rate of mice was observed, and the anti-inflammatory effect of 2.4EDB on EAM was preliminarily evaluated.After the second immunization, the mice were treated with 4EDB by gavage and the mice were killed after 21 days. The myocardial tissue was taken out, HE staining and Anabaena staining were performed, and the RNA was extracted.Pathological scores were obtained by HE staining, fibrosis was evaluated by Anabaena red staining, and the expression levels of IL-l 尾 -IL-6 IL-17A and TGF- 尾 in myocardial tissue were detected by real-time quantitative fluorescence PCR(quantitative real-time PCR qRT-PCR.Mouse eyeball blood was collected by centrifugation and supernatant was collected by centrifugation. The expression level of IL-l 尾 -IL-6, IL-17, IL-4 and IFN- 緯 in serum of mice was detected by enzyme-linked immunosorbent assay (Elisa) and enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay). The mechanism of EAM treated by 3.4EDB was discussed.The effect of 4EDB on macrophage apoptosis and the effect of 4EDB on macrophage chemotaxis were detected by flow cytometry (FCM) and Transwell assay respectively.The result is 1: 1.Sinomenine derivative 4EDBs were successfully prepared with purity of 99.59.The survival rate of mice treated with 4EDB was higher than that of sinomenine treated mice by sepsis model experiment.And the anti-inflammatory effect of 5 mg/kg 4EDB treatment group was better than that of 2.4EDB treatment of EAM model mice.We found that 5 mg/kg 4EDB could alleviate the progress of EAM. Elisa and qRT-PCR results showed that 4EDB could significantly reduce the content of IL-17AfIL-l 尾 -IL-6 in the serum of EAM mice, and the results of Tianlang Anabarene staining showed that 4EDB could alleviate the degree of myocardial fibrosis in EAM mice.The results of flow cytometry and Transwell showed that 10 nmol/mL or less 4EDB could not only induce the apoptosis of RAW264.7 cells, but also inhibit the migration of RAW264.7 cells.Conclusion 1.A new sinomenine derivative, 4EDBs, was synthesized and its purity was 99.590.Compared with sinomenine, 4EDB had a better anti-inflammatory effect. 2.4EDB could down-regulate the level of IL-l 尾 -IL-6IL-17A and alleviate the progression of myocardial fibrosis.The therapeutic effect of 3.4 EDB on EAM in EAM mice may be mediated by inducing myocardial macrophage apoptosis and inhibiting monocyte / macrophage migration.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R542.21

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