一种新型抗炎多肽的应用研究

发布时间：2019-01-24 22:23

【摘要】：目的： NLRC5是一种具有强烈抗炎效应的分子。本文通过生物信息学的方法，试图筛选出NLRC5特异性多肽，并研究其在细胞水平和动物水平的抗炎效应。方法： 1.通过生物信息学的方法筛选得到多肽LS22 运用生物信息学原理,首先将筛选的序列用SMART软件进行多序列比对,找出NLRC5蛋白的的保守序列。使用clustalx软件对NLRCx蛋白的序列进行同源比对，采用CLC Protein WorκBench软件对NLRC5蛋白的亲水性（Hydrophilicity）、抗原性（Anti-genicity）等物理特性进行分析；最后从LRR序列中筛选出LS22。 2. LS22多肽体外抗炎效应研究通过MTS实验检测LS22多肽对细胞活力的影响。通过脂多糖(LPS)分别刺激小鼠巨噬细胞Raw264.7，人脐静脉上皮细胞HUVEC，人单核细胞THP-1来建立体外炎症模型。通过ELISA和Q-PCR试验分别检测加入不同浓度的LS22多肽对于LPS诱导的炎症反应的影响。并采用LPS诱导的趋化实验和TNF-α诱导的粘附实验来检测LS22多肽是否也影响了细胞趋化反应和粘附能力。 3. LS22多肽体内抗炎效应研究采用LPS单次足垫注射法来诱导大鼠内毒素诱导的葡萄眼（EIU）。将内毒素溶于PBS中，使溶液浓度为2g/L，，将100μL内毒素分别注射于实验组大鼠的双后足底部；正常对照组后足底部仅注射100μL生理盐水。随后按分组将地塞米松、混杂序列肽、不同浓度LS22往大鼠双眼玻璃体腔内注射10μL；空白对照组和LPS组大鼠玻璃体腔内仅注射PBS10μL。于注射前及注射后第24h进行裂隙灯检查，详细记求临床体征的改变。通过大鼠眼球活组织镜检，组织切片，EIU疾病打分，房水中浸润细胞数目，房水总蛋白及细胞因子等不同的疾病特征，来检测LS22体内抗炎效应。 4. LS22多肽抗炎机制研究通过荧光素酶报告基因实验检测LS22多肽对于LPS诱导的NF-kb信号通路活化的影响。通过共聚焦实验和免疫印记试验，检测LS22多肽在RAW264.7细胞内的作用位点及其对于LPS诱导的转录因子p65磷酸化及入核的影响。结果： 1.多肽序列通过上述方法，我们找到了与NLRC5的LRR结构域类似的多肽LS22。其氨基酸序列为LDLSHNSISQESALYLLETLPS。 2. LS22多肽体外抗炎效应研究结果显示，LS22多肽对于细胞活力无影响。不论从蛋白水平还是mRNA水平，对于检测的三种细胞系，50μM、100μM的LS22多肽与阳性对照和混杂序列多肽组相比都，并且有剂量依赖关系，1μM无影响。LS22多肽抑制了LPS诱导的趋化反应，但对TNF-α诱导的细胞粘附能力无影响。 3. LS22多肽体内抗炎效应研究结果显示，LS22多肽显著抑制了临床疾病特征。前房炎症以及充血程度明显降低，疾病打分减少。病理组织切片结果显示LS22多肽显著抑制了虹膜睫状体部位和玻璃体后部视网膜区域炎症细胞浸润情况。房水中浸润细胞数目减少，房水总蛋白降低，房水中炎症因子TNF-α和IL-6水平也明显下降。 4. LS22多肽抗炎机制研究 LS22多肽抑制了LPS诱导的NF-kb荧光素酶报告基因的活化。共聚焦实验和免疫印记实验显示LS22多肽抑制p65的磷酸化及入核，但是对总p65无影响。结论： 1.通过生物信息学手段，预测得到LS22多肽 2. LS22多肽在体外具有抑制炎症的功能，并且具有剂量依赖效应 3. LS22多肽具有抑制大鼠葡萄膜炎及体内炎症的功能 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
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R773.9

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