本文選題：內(nèi)毒素　切入點：mEBP9　出處：《第三軍醫(yī)大學》2012年碩士論文

【摘要】：革蘭氏陰性桿菌外膜的主要成分內(nèi)毒素(lipopolysaccharide, LPS)是臨床上導致全身炎癥反應(yīng)綜合征(systemic inflammatory response syndrome,SIRS)和膿毒癥(sepsis)的主要原因。目前SIRS或者膿毒癥仍然具有較高的發(fā)病率和死亡率，現(xiàn)階段防治革蘭氏陰性桿菌引起的SIRS或者膿毒癥主要依賴于抗生素控制感染，但對內(nèi)毒素血癥(Endotoxemia)的療效并不理想。 脂多糖結(jié)合蛋白(lipopolysaccharide binding protein,LBP)具有放大LPS毒性的作用。LBP的氨基端含有特異性LPS結(jié)合位點；LBP的第91-108位氨基酸序列是已知的結(jié)合LPS能力很強的氨基酸序列(Endotoxin binding peptide，EBP)。在本課題組前期工作中，為增強EBP的拮抗LPS生物活性，通過對EBP的結(jié)構(gòu)進行改建，從11條內(nèi)毒素結(jié)合肽突變體(mutant of EBP，mEBP)中篩選出拮抗LPS作用較強的第9條內(nèi)毒素結(jié)合肽突變體(mEBP9)。鑒于普通多肽在體內(nèi)容易被降解，生物半衰期較短，而經(jīng)聚乙二醇修飾的多肽在體內(nèi)比未經(jīng)修飾的多肽可能具有較高的穩(wěn)定性和較好的保護效應(yīng)；本研究在mEBP9的氨基端或者羧基端引入一個半胱氨酸(mEBP9-SH)，通過mEBP9-SH上的巰基加成到mPEG-MAL(甲氧基聚乙二醇馬來酰亞胺)的雙鍵上，實現(xiàn)對mEBP9進行聚乙二醇定點修飾。 我們課題組通過生物合成途徑已經(jīng)獲得mEBP9、 PEG-NH-mEBP9以及PEG-COOH-mEBP9，觀察PEG-NH-mEBP9和PEG-COOH-mEBP9的拮抗LPS活性并篩選出拮抗LPS效應(yīng)更強的PEG-NH-mEBP9；在此基礎(chǔ)上比較PEG-NH-mEBP9與mEBP9的體內(nèi)外拮抗LPS活性。主要研究結(jié)果如下： 1. CCK-8法檢測結(jié)果顯示1、5、10、20μmol/L的mEBP9、PEG-NH-mEBP9和PEG-COOH-mEBP9對RAW264.7細胞均無可見的細胞毒性。 2. PEG-NH-mEBP9抑制LPS誘導的TAL活化效應(yīng)明顯強于同濃度的PEG-COOH-mEBP9(P＜0.01)；PEG-NH-mEBP9抑制LPS誘導的RAW264.7細胞分泌TNF-α和IL-6作用優(yōu)于等濃度的PEG-COOH-mEBP9。 3.10μmol/L的PEG-NH-mEBP9或mEBP9在-20℃放置30天后抑制LPS誘導的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℃環(huán)境放置30天后抑制LPS誘導的TAL活化能力大于等濃度的PEG-NH-mEBP9；10μmol/L的PEG-NH-mEBP9在37℃環(huán)境放置30天后抑制LPS誘導的TAL活化作用明顯強于同濃度的mEBP9(P＜0.05)。LPS誘導RAW264.7細胞3h、6h、12h后，5μmol/L的mEBP9抑制RAW264.7細胞分泌TNF-α效應(yīng)均強于同一時相點同濃度的PEG-NH-mEBP9；LPS刺激RAW264.7細胞24h、36h、48h后，5μmol/L的PEG-NH-mEBP9的抑制RAW264.7細胞分泌TNF-α能力反而都強于同一時相點等濃度的mEBP9。 4. PEG-NH-mEBP9與mEBP9體外拮抗LPS生物活性比較：mEBP9抑制LPS誘導的TAL活化效應(yīng)強于同濃度的PEG-NH-mEBP9；mEBP9抑制LPS誘導的RAW264.7細胞分泌TNF-α和IL-6作用明顯優(yōu)于等濃度的PEG-NH-mEBP9(P＜0.05)。 5. PEG-NH-mEBP9和mEBP9體內(nèi)拮抗LPS效應(yīng)比較：(1)提高模型小鼠生存率：“LPS/D-GalN”組48h內(nèi)全部死亡，“mEBP9+LPS/D-GalN”組和“PEG-NH-mEBP9+LPS/D-GalN”組48h生存率分別為為40%和60%。(2)減少血漿TNF-α和IL-6水平：LPS/D-GalN處理后90min，PEG-NH-mEBP9和mEBP9都可以明顯抑制小鼠體內(nèi)血漿TNF-α/IL-6分泌(P＜0.01)，且PEG-NH-mEBP9拮抗LPS的效應(yīng)顯著強于mEBP9(P＜0.05)。(3)減少血清ALT和AST水平：LPS/D-GalN處理后6h，PEG-NH-mEBP9和mEBP9都可以明顯抑制小鼠體內(nèi)血清ALT/AST活性(P＜0.01)，且PEG-NH-mEBP9拮抗LPS活性明顯強于mEBP9(P＜0.05)。(4)減輕小鼠肝臟病理組織學改變：“LPS/D-GalN”組肝臟有明顯的病理學改變，包括肝細胞壞死，充血，肝組織結(jié)構(gòu)的破壞，肝竇內(nèi)大量的炎癥細胞聚集；“LPS/D-GalN+mEBP9”組肝臟組織壞死的區(qū)域和壞死程度均降低，而且炎癥細胞浸潤減少；“LPS/D-GalN+PEG-NH-mEBP9”組肝臟病理組織學改變程度比“LPS/D-GalN+mEBP9”組輕。 主要研究結(jié)論：PEG-NH-mEBP9拮抗LPS效應(yīng)優(yōu)于PEG-COOH-mEBP9；PEG-NH-mEBP9拮抗LPS的生物活性稍遜于mEBP9，，而PEG-NH-mEBP9的體外穩(wěn)定性優(yōu)于mEBP9；PEG-NH-mEBP9和mEBP9均可保護D-氨基半乳糖增敏的內(nèi)毒素血癥模型小鼠，且PEG-NH-mEBP9對模型小鼠的保護作用優(yōu)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 - 偽 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

本文編號：1692828