一種新型抗炎多肽的應(yīng)用研究
發(fā)布時間：2019-01-24 22:23
【摘要】：目的： NLRC5是一種具有強烈抗炎效應(yīng)的分子。本文通過生物信息學(xué)的方法，試圖篩選出NLRC5特異性多肽，并研究其在細胞水平和動物水平的抗炎效應(yīng)。 方法： 1.通過生物信息學(xué)的方法篩選得到多肽LS22 運用生物信息學(xué)原理,首先將篩選的序列用SMART軟件進行多序列比對,找出NLRC5蛋白的的保守序列。使用clustalx軟件對NLRCx蛋白的序列進行同源比對，采用CLC Protein WorκBench軟件對NLRC5蛋白的親水性（Hydrophilicity）、抗原性（Anti-genicity）等物理特性進行分析；最后從LRR序列中篩選出LS22。 2. LS22多肽體外抗炎效應(yīng)研究 通過MTS實驗檢測LS22多肽對細胞活力的影響。通過脂多糖(LPS)分別刺激小鼠巨噬細胞Raw264.7，人臍靜脈上皮細胞HUVEC，人單核細胞THP-1來建立體外炎癥模型。通過ELISA和Q-PCR試驗分別檢測加入不同濃度的LS22多肽對于LPS誘導(dǎo)的炎癥反應(yīng)的影響。并采用LPS誘導(dǎo)的趨化實驗和TNF-α誘導(dǎo)的粘附實驗來檢測LS22多肽是否也影響了細胞趨化反應(yīng)和粘附能力。 3. LS22多肽體內(nèi)抗炎效應(yīng)研究 采用LPS單次足墊注射法來誘導(dǎo)大鼠內(nèi)毒素誘導(dǎo)的葡萄眼（EIU）。將內(nèi)毒素溶于PBS中，使溶液濃度為2g/L，，將100μL內(nèi)毒素分別注射于實驗組大鼠的雙后足底部；正常對照組后足底部僅注射100μL生理鹽水。隨后按分組將地塞米松、混雜序列肽、不同濃度LS22往大鼠雙眼玻璃體腔內(nèi)注射10μL；空白對照組和LPS組大鼠玻璃體腔內(nèi)僅注射PBS10μL。于注射前及注射后第24h進行裂隙燈檢查，詳細記求臨床體征的改變。通過大鼠眼球活組織鏡檢，組織切片，EIU疾病打分，房水中浸潤細胞數(shù)目，房水總蛋白及細胞因子等不同的疾病特征，來檢測LS22體內(nèi)抗炎效應(yīng)。 4. LS22多肽抗炎機制研究 通過熒光素酶報告基因?qū)嶒灆z測LS22多肽對于LPS誘導(dǎo)的NF-kb信號通路活化的影響。通過共聚焦實驗和免疫印記試驗，檢測LS22多肽在RAW264.7細胞內(nèi)的作用位點及其對于LPS誘導(dǎo)的轉(zhuǎn)錄因子p65磷酸化及入核的影響。 結(jié)果： 1.多肽序列 通過上述方法，我們找到了與NLRC5的LRR結(jié)構(gòu)域類似的多肽LS22。其氨基酸序列為LDLSHNSISQESALYLLETLPS。 2. LS22多肽體外抗炎效應(yīng)研究 結(jié)果顯示，LS22多肽對于細胞活力無影響。不論從蛋白水平還是mRNA水平，對于檢測的三種細胞系，50μM、100μM的LS22多肽與陽性對照和混雜序列多肽組相比都，并且有劑量依賴關(guān)系，1μM無影響。LS22多肽抑制了LPS誘導(dǎo)的趨化反應(yīng)，但對TNF-α誘導(dǎo)的細胞粘附能力無影響。 3. LS22多肽體內(nèi)抗炎效應(yīng)研究 結(jié)果顯示，LS22多肽顯著抑制了臨床疾病特征。前房炎癥以及充血程度明顯降低，疾病打分減少。病理組織切片結(jié)果顯示LS22多肽顯著抑制了虹膜睫狀體部位和玻璃體后部視網(wǎng)膜區(qū)域炎癥細胞浸潤情況。房水中浸潤細胞數(shù)目減少，房水總蛋白降低，房水中炎癥因子TNF-α和IL-6水平也明顯下降。 4. LS22多肽抗炎機制研究 LS22多肽抑制了LPS誘導(dǎo)的NF-kb熒光素酶報告基因的活化。共聚焦實驗和免疫印記實驗顯示LS22多肽抑制p65的磷酸化及入核，但是對總p65無影響。 結(jié)論： 1.通過生物信息學(xué)手段，預(yù)測得到LS22多肽 2. LS22多肽在體外具有抑制炎癥的功能，并且具有劑量依賴效應(yīng) 3. LS22多肽具有抑制大鼠葡萄膜炎及體內(nèi)炎癥的功能 4. LS22多肽能抑制p65磷酸化及入核
[Abstract]:Purpose: NLRC5 is a fraction with a strong anti-inflammatory effect This paper, by means of bioinformatics, tries to screen the NLRC5-specific polypeptide and to study its anti-inflammatory effect at cell level and animal level. It is to be. Method: 1. Screening by means of bioinformatics In the application of the principle of bioinformatics, the polypeptide LS22 firstly uses the SMART software to carry out the multi-sequence comparison, and the NLRC5 egg is found out. The conserved sequence of NLRCx protein was compared with the sequence of NLRCx protein by using the clusalx software. The physical properties of NLRC5 protein, such as Hydrophilicity, Anti-genicity, were analyzed by using the CLC Protein Wor Benoch software, and finally from the LRR sequence. LS22. 2. LS22 filtered Study on the anti-inflammatory effect of polypeptide in vitro by MTS assay 2. The effect of the polypeptide on the cell viability. The mouse macrophage Raw264.7, human umbilical vein epithelial cell HUVEC and human monocyte THP were stimulated by lipopolysaccharide (LPS), respectively.-1 to establish an in vitro inflammatory model. LS22 polypeptides of different concentrations were detected for the LP by ELISA and Q-PCR assays, respectively. The effect of S-induced inflammatory response was investigated and the LPS-induced chemotaxis and TNF-induced adhesion experiments were used to detect whether the LS22 polypeptide has also been affected. Cell chemotaxis and adhesion. 3. The study of the anti-inflammatory effect of LS22 polypeptide in vivo was induced by LPS single-term foot pad injection method. The endotoxin-induced grape eye (EIU) was dissolved in PBS, the concentration of the solution was 2g/ L, and the 100 & mu; L endotoxin was injected into the bottom of the double-foot foot of the experimental group rats respectively; and after the normal control group, 100. m u.L of physiological saline was injected at the bottom of the foot. Dexamethasone, the mixed sequence peptide and the LS22 at different concentrations were then injected into the vitreous cavity of the rat with 10. m u.L in the group. In the blank control group and the LPS group, only PBS10.mu. L was injected into the vitreous cavity of the rat. The fracture was performed before and after the injection. The changes of clinical signs were found in the light examination and detailed records. The different diseases, such as the number of cells in the room, the total protein of the aqueous humor and the cytokines, were scored by the microscopic examination of the eye biopsy of the rat, the tissue section, the EIU disease, the number of infiltration cells in the room water, the total protein of the aqueous humor, and the cytokines. Sign, to detect the anti-inflammatory effect in LS22 The study of the anti-inflammatory mechanism of the polypeptide of the. 4. LS22 polypeptide through the luciferase reporter gene experiment to detect the LS22 polypeptide for L Effect of PS-induced activation of NF-kb signal pathway. The role of LS22 polypeptide in RAW264.7 cells and its effect on LPS were tested by co-focusing and imprints. induced transcription factor p6 5. Effect of Phosphorylation and Nucleation. Results: 1. The polypeptide sequence was obtained by the method described above. A polypeptide LS22 similar to the LRR domain of the NLRC5 is found. The sequence is LDLSHNSISQESA LYLLETLPS. 2.LS22 polypeptide body The results of the external anti-inflammatory effect show that the LS22 polypeptide has no effect on the cell viability. There was no effect on 1. m u.M and 1. m no effect on the LS22 polypeptide compared to the mixed-sequence polypeptide group. PS-induced chemotaxis, but for TNF-There was no effect on the ability of the cells to adhere to the cells. The results of the anti-inflammatory effect in the peptides show that the LS22 polypeptide The clinical features of the disease were significantly inhibited. The inflammation of the anterior chamber and the degree of hyperemia were significantly reduced, and the score of the disease was reduced. The results of the pathological tissue slice showed that the LS22 Polypeptides significantly inhibited the infiltration of inflammatory cells in the iris-ciliary body and in the posterior retinal region of the vitreous. The number of infiltration cells in the room was reduced. and the total protein of the aqueous humor is reduced, and the inflammation in the room water The levels of TNF-1 and IL-6 were also significantly decreased. The mechanism study the activation of the LPS-induced NF-kb luciferase reporter gene by the LS22 polypeptide. immunoprinting The results show that the LS22 polypeptide inhibits the phosphorylation and the in-in of p65. Nuclear, but no effect on total p65. Conclusion: 1. Bioinformatics and the LS22 polypeptide is predicted to have the LS22 polypeptide in vitro. the function of inhibiting inflammation and having a dose-dependent effect
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R773.9
[Abstract]:Purpose: NLRC5 is a fraction with a strong anti-inflammatory effect This paper, by means of bioinformatics, tries to screen the NLRC5-specific polypeptide and to study its anti-inflammatory effect at cell level and animal level. It is to be. Method: 1. Screening by means of bioinformatics In the application of the principle of bioinformatics, the polypeptide LS22 firstly uses the SMART software to carry out the multi-sequence comparison, and the NLRC5 egg is found out. The conserved sequence of NLRCx protein was compared with the sequence of NLRCx protein by using the clusalx software. The physical properties of NLRC5 protein, such as Hydrophilicity, Anti-genicity, were analyzed by using the CLC Protein Wor Benoch software, and finally from the LRR sequence. LS22. 2. LS22 filtered Study on the anti-inflammatory effect of polypeptide in vitro by MTS assay 2. The effect of the polypeptide on the cell viability. The mouse macrophage Raw264.7, human umbilical vein epithelial cell HUVEC and human monocyte THP were stimulated by lipopolysaccharide (LPS), respectively.-1 to establish an in vitro inflammatory model. LS22 polypeptides of different concentrations were detected for the LP by ELISA and Q-PCR assays, respectively. The effect of S-induced inflammatory response was investigated and the LPS-induced chemotaxis and TNF-induced adhesion experiments were used to detect whether the LS22 polypeptide has also been affected. Cell chemotaxis and adhesion. 3. The study of the anti-inflammatory effect of LS22 polypeptide in vivo was induced by LPS single-term foot pad injection method. The endotoxin-induced grape eye (EIU) was dissolved in PBS, the concentration of the solution was 2g/ L, and the 100 & mu; L endotoxin was injected into the bottom of the double-foot foot of the experimental group rats respectively; and after the normal control group, 100. m u.L of physiological saline was injected at the bottom of the foot. Dexamethasone, the mixed sequence peptide and the LS22 at different concentrations were then injected into the vitreous cavity of the rat with 10. m u.L in the group. In the blank control group and the LPS group, only PBS10.mu. L was injected into the vitreous cavity of the rat. The fracture was performed before and after the injection. The changes of clinical signs were found in the light examination and detailed records. The different diseases, such as the number of cells in the room, the total protein of the aqueous humor and the cytokines, were scored by the microscopic examination of the eye biopsy of the rat, the tissue section, the EIU disease, the number of infiltration cells in the room water, the total protein of the aqueous humor, and the cytokines. Sign, to detect the anti-inflammatory effect in LS22 The study of the anti-inflammatory mechanism of the polypeptide of the. 4. LS22 polypeptide through the luciferase reporter gene experiment to detect the LS22 polypeptide for L Effect of PS-induced activation of NF-kb signal pathway. The role of LS22 polypeptide in RAW264.7 cells and its effect on LPS were tested by co-focusing and imprints. induced transcription factor p6 5. Effect of Phosphorylation and Nucleation. Results: 1. The polypeptide sequence was obtained by the method described above. A polypeptide LS22 similar to the LRR domain of the NLRC5 is found. The sequence is LDLSHNSISQESA LYLLETLPS. 2.LS22 polypeptide body The results of the external anti-inflammatory effect show that the LS22 polypeptide has no effect on the cell viability. There was no effect on 1. m u.M and 1. m no effect on the LS22 polypeptide compared to the mixed-sequence polypeptide group. PS-induced chemotaxis, but for TNF-There was no effect on the ability of the cells to adhere to the cells. The results of the anti-inflammatory effect in the peptides show that the LS22 polypeptide The clinical features of the disease were significantly inhibited. The inflammation of the anterior chamber and the degree of hyperemia were significantly reduced, and the score of the disease was reduced. The results of the pathological tissue slice showed that the LS22 Polypeptides significantly inhibited the infiltration of inflammatory cells in the iris-ciliary body and in the posterior retinal region of the vitreous. The number of infiltration cells in the room was reduced. and the total protein of the aqueous humor is reduced, and the inflammation in the room water The levels of TNF-1 and IL-6 were also significantly decreased. The mechanism study the activation of the LPS-induced NF-kb luciferase reporter gene by the LS22 polypeptide. immunoprinting The results show that the LS22 polypeptide inhibits the phosphorylation and the in-in of p65. Nuclear, but no effect on total p65. Conclusion: 1. Bioinformatics and the LS22 polypeptide is predicted to have the LS22 polypeptide in vitro. the function of inhibiting inflammation and having a dose-dependent effect
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R773.9
【共引文獻】
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