内毒素结合肽突变体（mEBP9）的聚乙二醇定点修饰及其稳定性和拮抗内毒素效应研究

发布时间：2018-03-31 23:00

  本文选题：内毒素　切入点：mEBP9　出处：《第三军医大学》2012年硕士论文

【摘要】：革兰氏阴性杆菌外膜的主要成分内毒素(lipopolysaccharide, LPS)是临床上导致全身炎症反应综合征(systemic inflammatory response syndrome,SIRS)和脓毒症(sepsis)的主要原因。目前SIRS或者脓毒症仍然具有较高的发病率和死亡率，现阶段防治革兰氏阴性杆菌引起的SIRS或者脓毒症主要依赖于抗生素控制感染，但对内毒素血症(Endotoxemia)的疗效并不理想。 脂多糖结合蛋白(lipopolysaccharide binding protein,LBP)具有放大LPS毒性的作用。LBP的氨基端含有特异性LPS结合位点；LBP的第91-108位氨基酸序列是已知的结合LPS能力很强的氨基酸序列(Endotoxin binding peptide，EBP)。在本课题组前期工作中，为增强EBP的拮抗LPS生物活性，通过对EBP的结构进行改建，从11条内毒素结合肽突变体(mutant of EBP，mEBP)中筛选出拮抗LPS作用较强的第9条内毒素结合肽突变体(mEBP9)。鉴于普通多肽在体内容易被降解，生物半衰期较短，而经聚乙二醇修饰的多肽在体内比未经修饰的多肽可能具有较高的稳定性和较好的保护效应；本研究在mEBP9的氨基端或者羧基端引入一个半胱氨酸(mEBP9-SH)，通过mEBP9-SH上的巯基加成到mPEG-MAL(甲氧基聚乙二醇马来酰亚胺)的双键上，实现对mEBP9进行聚乙二醇定点修饰。 我们课题组通过生物合成途径已经获得mEBP9、 PEG-NH-mEBP9以及PEG-COOH-mEBP9，观察PEG-NH-mEBP9和PEG-COOH-mEBP9的拮抗LPS活性并筛选出拮抗LPS效应更强的PEG-NH-mEBP9；在此基础上比较PEG-NH-mEBP9与mEBP9的体内外拮抗LPS活性。主要研究结果如下： 1. CCK-8法检测结果显示1、5、10、20μmol/L的mEBP9、PEG-NH-mEBP9和PEG-COOH-mEBP9对RAW264.7细胞均无可见的细胞毒性。 2. PEG-NH-mEBP9抑制LPS诱导的TAL活化效应明显强于同浓度的PEG-COOH-mEBP9(P＜0.01)；PEG-NH-mEBP9抑制LPS诱导的RAW264.7细胞分泌TNF-α和IL-6作用优于等浓度的PEG-COOH-mEBP9。 3.10μmol/L的PEG-NH-mEBP9或mEBP9在-20℃放置30天后抑制LPS诱导的TAL活化能力大于在37℃放置30天后的PEG-NH-mEBP9或mEBP9；10μmol/L的mEBP9在-20℃环境放置30天后抑制LPS诱导的TAL活化能力大于等浓度的PEG-NH-mEBP9；10μmol/L的PEG-NH-mEBP9在37℃环境放置30天后抑制LPS诱导的TAL活化作用明显强于同浓度的mEBP9(P＜0.05)。LPS诱导RAW264.7细胞3h、6h、12h后，5μmol/L的mEBP9抑制RAW264.7细胞分泌TNF-α效应均强于同一时相点同浓度的PEG-NH-mEBP9；LPS刺激RAW264.7细胞24h、36h、48h后，5μmol/L的PEG-NH-mEBP9的抑制RAW264.7细胞分泌TNF-α能力反而都强于同一时相点等浓度的mEBP9。 4. PEG-NH-mEBP9与mEBP9体外拮抗LPS生物活性比较：mEBP9抑制LPS诱导的TAL活化效应强于同浓度的PEG-NH-mEBP9；mEBP9抑制LPS诱导的RAW264.7细胞分泌TNF-α和IL-6作用明显优于等浓度的PEG-NH-mEBP9(P＜0.05)。 5. PEG-NH-mEBP9和mEBP9体内拮抗LPS效应比较：(1)提高模型小鼠生存率：“LPS/D-GalN”组48h内全部死亡，“mEBP9+LPS/D-GalN”组和“PEG-NH-mEBP9+LPS/D-GalN”组48h生存率分别为为40%和60%。(2)减少血浆TNF-α和IL-6水平：LPS/D-GalN处理后90min，PEG-NH-mEBP9和mEBP9都可以明显抑制小鼠体内血浆TNF-α/IL-6分泌(P＜0.01)，且PEG-NH-mEBP9拮抗LPS的效应显著强于mEBP9(P＜0.05)。(3)减少血清ALT和AST水平：LPS/D-GalN处理后6h，PEG-NH-mEBP9和mEBP9都可以明显抑制小鼠体内血清ALT/AST活性(P＜0.01)，且PEG-NH-mEBP9拮抗LPS活性明显强于mEBP9(P＜0.05)。(4)减轻小鼠肝脏病理组织学改变：“LPS/D-GalN”组肝脏有明显的病理学改变，包括肝细胞坏死，充血，肝组织结构的破坏，肝窦内大量的炎症细胞聚集；“LPS/D-GalN+mEBP9”组肝脏组织坏死的区域和坏死程度均降低，而且炎症细胞浸润减少；“LPS/D-GalN+PEG-NH-mEBP9”组肝脏病理组织学改变程度比“LPS/D-GalN+mEBP9”组轻。 主要研究结论：PEG-NH-mEBP9拮抗LPS效应优于PEG-COOH-mEBP9；PEG-NH-mEBP9拮抗LPS的生物活性稍逊于mEBP9，，而PEG-NH-mEBP9的体外稳定性优于mEBP9；PEG-NH-mEBP9和mEBP9均可保护D-氨基半乳糖增敏的内毒素血症模型小鼠，且PEG-NH-mEBP9对模型小鼠的保护作用优于mEBP9。
[Abstract]:Endotoxin ( LPS ) is the main cause of systemic inflammatory response syndrome ( systemic inflammatory response syndrome ) and sepsis ( sepsis ) . At present , there is still a high morbidity and mortality rate . At the present stage , the prevention and treatment of systemic inflammatory response syndrome caused by gram - negative bacilli is mainly dependent on antibiotic - controlled infection , but it is not ideal for endotoxaemia .

lipopolysaccharide binding protein ( lbp ) has the function of amplifying lps toxicity .
The amino acid sequence of the amino acid sequence 91 - 108 is known to bind to an amino acid sequence ( EBP ) which has a strong ability to bind LPS . In the previous work of the study group , to enhance the antagonistic LPS bioactivity of EBP , the article 9 endotoxin - binding peptide mutant ( mEBP9 ) was screened from 11 endotoxin - binding peptide mutants ( mutant of EBP , mEBP ) .
In this study , a cysteine ( mEB9 - SH ) was introduced at the amino end or carboxyl terminal of mEBP9 , and the thiol group on mEB9 - SH was added to the double bond of mPEG - MAL ( methoxypolyethylene glycol maleimide ) .

The antagonistic LPS activity of PEG - NH - mEB9 and PEG - COOH - mEBP9 , PEG - NH - mEBP9 and PEG - COOH - mEBP9 , and PEG - NH - mEBP9 , which antagonized LPS effect , were obtained by means of biosynthetic pathway .
On the basis of this , the activity of LPS was antagonized by PEG - NH - mEBP9 and mEBP9 . The results were as follows :

1 . The results of CCK - 8 assay showed that 1 , 5 , 10 , 20 渭mol / L mEB9 , PEG - NH - mEB9 and PEG - COOH - mEBP9 had no cytotoxicity on RAW264.7 cells .

2 . PEG - NH - mEBP9 inhibited LPS - induced activation of TAL significantly stronger than PEG - COOH - mEBP9 ( P < 0.01 ) .
PEG - NH - mEBP9 inhibited LPS - induced TNF - 伪 and IL - 6 secretion in RAW264.7 cells .

3 . 10 渭mol / L PEG - NH - mEBP9 or mEBP9 inhibited LPS - induced TAL activation at -20 鈩

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