【作者】 王云龙；

【导师】 陈保生；

【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 高密度脂蛋白很早以前就被证明能够中和内毒素的毒性。载脂蛋白A-I是高密度脂蛋白中主要的蛋白成分,因此其在中和内毒素毒性方面上发挥了至关重要的作用。根据载脂蛋白A-I的两个天然突变体的突变位点及Edmunson轮,我们课题组利用PET表达系统已经成功构建出包括天然突变体ApoA-Imilano在内的七个半胱氨酸突变体,分别为A-I（S52C）、A-I（N74C）、A-I（K107C）、A-I（G129C）、A-I_M（R173C）、A-I（K195C）、A-I（S228C）。实验证明,这些突变体无论是在二级结构还是体外功能上都不尽相同,这就增加了突变后会影响其体内功能的可能性。实验目的:本研究对野生型apoA-I和分别发生在apoA-I多肽链7个α螺旋片段上的半胱氨酸突变体结合脂质后生成的八种重组高密度脂蛋白（rHDLwt,rHDL52,rHDL74,rHDL107,rHDL129,rHDL173,rHDL195和rHDL228）的抗炎性能做了比较,以观察这些特定的半胱氨酸突变体的结构和功能之间是否存在某些必然的联系。我们希望这些问题的解答能够对我们更深入、更全面的认识高密度脂蛋白抗炎保护特性的机制有所帮助。实验方法:利用pET原核表达系统对野生型及突变的apoA-I进行蛋白质表达,Ni²⁺亲和柱进行目的蛋白纯化,获得多肽链C-末端带有6个组氨酸标签（6×His tag）的高纯度蛋白（纯度＞90%）,再经过去内毒素处理,最后利用胆酸盐透析方法做成重组的高密度脂蛋白用于下面的体内功能实验。我们利用尾静脉注射脂多糖来诱导小鼠内毒素血症模型的生成,并以其肛温升高0.5度作为标准,然后再注射重组的高密度脂蛋白。同时为了更加准确的评估这几种重组高密度脂蛋白的抗炎性能,我们选择了两个时间点加以分析,以注射脂多糖为计时点,检测其注射后3h和24h的重组高密度脂蛋白的治疗效果,主要通过ELISA试剂盒检测血清中的促炎症因子的水平,并对注射LPS 24h后的小鼠的肺组织做了常规的病理切片分析。为了进一步探索高密度脂蛋白的抗炎机制,我们分析了利用野生型aDoA-I及其半胱氨酸突变体结合脂质后形成的重组高密度脂蛋白的成份,即:脂质与apoA-I或其半胱氨酸突变体的比率。实验结果:内毒素血症小鼠模型的构建:BALB/c小鼠尾静脉注射LPS后,大约11.5min,即可检测到其肛温升高0.5℃,从而可以判定内毒素血症小鼠模型构建成功。血清中炎症因子的检测:LPS注射后24h的结果:LPS注射24h后,经ELISA试剂盒检测,结果显示:与野生型A-I构建的重组高密度脂蛋白rHDLwt相比,rHDL74和rHDL52更能显著降低小鼠血清中TNF-α的水平,而且,经过rHDL74治疗后,其TNF-α的水平已经降低到正常水平。与此相反的是,经rHDL195及rHDL228处理的小鼠,其TNF-α水平与只注射LPS的对照组相比不但没有降低,反而升高。另外经过rHDLwt处理的小鼠,其TNF-α水平与LPS对照组相比,也明显的降低。同时,结果也显示,rHDLwt及其它半胱氨酸突变体构成的重组高密度脂蛋白（rHDL228除外）能显著降低LPS诱导产生的IL-1β水平,而且,与rHDLwt相比,rHDL74和rHDL52更能有效的降低IL-1β水平。除此之外,结果还显示,rHDL74能显著降低IL-6的水平,相反,rHDL52及rHDL107却能导致IL-6水平的升高。LPS注射3h的结果:与LPS注射24h结果相似,与只注射LPS的对照组相比,rHDL74、rHDL52以及rHDLwt治疗后的小鼠有较低的TNF-α水平,但其它重组高密度脂蛋白治疗后的小鼠没有明显的变化。同时,rHDL74、rHDL52以及rHDL107能降低IL-1β的水平,其它重组高密度脂蛋白对于IL-1β的水平没有明显的影响。另外,没有任何一种重组高密度脂蛋白能显著影响IL-6的水平。肺组织常规病理切片H&E染色分析:结果显示,只注射LPS的对照组,其肺组织有明显的病理学变化,包括:1)充血;2)肺间质组织增厚;3)白细胞浸润,包括单核细胞和中性粒细胞。而经过rHDLwt治疗后的小鼠的肺组织切片只显示了较轻的病理学变化,比如:较轻的肺间质组织增厚等等。经rHDL74治疗后的肺组织切片强力支持其具有保护肺组织抵抗LPS诱导的肺损伤,其H&E染色结果非常接近只注射生理盐水的对照组的染色结果,基本上没有明显的病理学变化。然而,rHDL228处理过的小鼠的肺组织切片却显示出相对于只注射LPS的对照组更严重的病理学变化。经其它几种重组高密度脂蛋白处理后,小鼠肺组织的病理学变化都明显比只注射LPS组的小鼠的肺组织要轻一些,显示除了rHDL228外,其它几种高密度脂蛋白都能发挥一定的抗炎作用。重组高密度脂蛋白成分的分析:rHDL74中的DPPC与apoA-I的质量比率要比rHDLwt中的高（1.93±0.06 vs.1.75±0.11）,相反rHDL228是几种重组高密度脂蛋白中比率最低的,与rHDLwt相比,rHDL228:（0.82±0.05 vs.1.75±0.11,P＜0.05）;rHDL107（1.49±0.12 vs.1.75±0.11,P＜0.05）and rHDL129（1.47±0.04 vs.1.75±0.11,P＜0.05）,这三种高密度脂蛋白中的DPPC与apoA-I的比率与rHDLwt相比都有所降低。其它重组高密度脂蛋白未见明显的差异。结论:1.rHDL74及rHDL52比rHDLwt更能有效的降低炎症因子TNF-α和IL-1β的水平。2.rHDL74比rHDLwt更能有效的降低炎症因子IL-6的水平。3.rHDL74及rHDL52能够比rHDLwt更有效的保护肺组织抵抗LPS诱导的损伤。4.rHDL228不但没有任何抗炎作用,相反它还能加剧炎症反应,增加LPS诱导的肺损伤。5.rHDL74中所含有的DPPC要比rHDLwt高,相反,rHDL228中的DPPC是这几种重组高密度脂蛋白中最低的。总之,半胱氨酸突变体不仅对apoA-I的二级结构和体外功能造成了一定的改变,而且还对其体内中和LPS的抗炎功能产生了巨大的影响,基于rHDL74在本实验中的保护作用,它可能会成为临床上极具潜力的治疗内毒素引起的败血症的基因工程药物。更多还原

【Abstract】 High density lipoprotein（HDL） has been shown able to neutralize the toxicity of the lipopolysaccharide（LPS）.ApoA-I is the principal protein constituent in HDL,and it is the key factor in the neutralization of the lipopolysaccharide.According to the mutant site of two natural mutants:ApoA-Imilano,ApoA-Ipairs and Edmunson wheels,our lab have succeeded in reconstituting seven ApoA-I cysteine mutants including ApoA-Imilano（A-I（S52C）、A-I（N74C）、A-I（K107C）、A-I（G129C）、A-I_M（R173C）、A-I（K195C）、A-I（S228C））,and the results showed that these mutants had different structural features or biological activities,suggesting the potential influence of the in vivo function of these mutations.AIM:In this study,we compared the anti-inflammation properties of wild-type apoA-I （wtapoA-I） and its 7 cysteine substitution mutants in lipid bound state.Our aim is to explore how the replacement of cysteine influences the structure and function of apoA-I,and whether there is a relationship between the structures and functions of mutants with specific cysteine mutant sites.We hope that our findings could shed some lights on the anti-inflammation mechanisms of HDL.METHODS:Both of wtapoA-I and the cysteine mutants were expressed with pET30b（+） as the expression vector and BL21（DE3） as the host bacterial,respectively.After purified by Ni²⁺ affinity chromatography,all of the proteins treated by Triton X-114 and Detoxi-GelTM Endotoxin Removing Gel in order to remove any residual endotoxin.Recombinant HDLs were prepared by using sodium cholate dialysis technique,then their in vivo anti-inflammation functions were examined by the septic mice which was induced by LPS.In order to assess the anti-inflammation of these rHDLs perfectly,we choose 3h and 24h two time point to measure the level of pro-inflammation cytokines in plasma by ELISA kits.Twenty four hours after LPS injection,lung was isolated from mice and fixed in 10%formaldehyde solution at room temperature,sectioned followed by stained with hematoxylin-eosin（H&E）.In order to further explore the anti-inflammation mechanism of rHDL,we examine the ratios of DPPC to apoA-I in these rHDLs.RESLUTS:The construction of endotoxemia mice modelLPS was injected into mice through tail vein.After the anal temperature increased 0.5℃after LPS injection,we can get the endotoxemia model.Detection the level of inflammatory cytokines in the plasma of the mice24h post LPS injection,compared to controls injected with rHDLwt （135.28±12.84pg/ml）,mice receiving either rHDL74 or rHDL52 exhibited significantly lower plasma levels of TNF-α（rHDL74:24.47±3.96pg/ml, P=0.002＜0.05 vs.rHDLwt;rHDL52:39.96±2.44pg/ml,P=0.009＜0.05 vs.rHDLwt）. The plasma concentration of TNF-αin rHDL74 treated mice was reduced down to the baseline level（24.16±1.63pg/ml）.Differently,mice treated with rHDL195 or rHDL228 had much higher plasma concentration of TNF-αcompared with LPS single injection groups（rHDL195:P=0.037＜0.05,rHDL228:P＜0.001,respectively）. Treatment with recombinant HDL containing wt apoA-I also resulted in a significant decrease of plasma TNF-α（135.28±12.84pg/ml,P=0.045＜0.05 vs.LPS group）. However,no statistical differences were observed in other groups of mice which were treated with rHDL107,rHDL129 or rHDL173 compared with LPS group.We also observed that the treatment of rHDLwt and all the other rHDL mutants except rHDL228 resulted in a decrease of plasma IL-1β（P＜0.001 vs.LPS）.Mice treated with rHDL74 and rHDL52 had significantly lower secretion of IL-1βthan those treated with rHDLwt,（rHDL74:P＜0.001;rHDL52:P=0.021＜0.05,respectively）. And other rHDLs（rHDL107,rHDL129,rHDL173 or rHDL195,P＜0.001,compared with LPS） also showed the capability of decreasing plasma IL-1βinduced by LPS injection.Besides these,we also found that treating mice with rHDL74 significantly attenuated plasma IL-6 production induced by in vivo LPS injection,compared with single LPS injection group（P=0.006＜0.05）;however,treatments with rHDL52 and rHDL107 led to an increase of plasma IL-6（rHDL52:P=0.009＜0.01.rHDL107: P=0.02＜0.05,respectively）.Similar to our observations at 24h after LPS injection,at 3h after LPS injection mice treated with rHDLwt,rHDL74 and rHDL52 had lower level of TNF-αthan those only received LPS injection（rHDLwt:P=0.049＜0.05,rHDL74:P=0.012＜0.05, rHDL52:P=0.048＜0.05,respectively）.No significant alteration was observed in other groups of mice treated with rHDL107,rHDL129,rHDL173 and rHDL228 compared with LPS group.Recombinant HDL74,rHDL52 and rHDL107 could decrease the level of IL-1β,compared with LPS group（rHDL74:P=0.003＜0.05,rHDL52: P=0.007＜0.05,rHDL107:P=0.034＜0.05）,while we did not see any significant effects of other rHDLs on reducing plasma IL-1β.None of the rHDLs showed a reduction of plasma IL-6 at 3h post LPS injection.Compared with 24h of LPS injection,the effect of rHDL on cytokine levels at 3 hours after LPS is biologically very small,suggesting a better neutralization of residual LPS in the circulation by rHDL injection for a longer time.Histological sections of lung tissueThe lung of mice only receiving LPS had significant pathological changes: 1)congestion.2) broadening of pulmonary interstitial tissue.3) leucocyte infiltration including monocytes and neutrophils,we observed that the mice treated with rHDLwt only exhibited slight pathological lung change,such as the moderate broadening of pulmonary interstitial tissue,compared with the saline group.In addition,the ability of rHDL74 to protect lung against LPS induced injury in this septic mice model is strongly supported by its histological results,which were very close to the saline group,and there was almost no pathological change in the Mut74 group.However,in lung sections from mice treated with rHDL228,we could not see any effect of reducing histological tissue injury,in stead of the aggravation of pathological changes, compared with LPS group.In addition,the histological sections of the other four rHDLs all had many pathological changes compared with the saline group.The weight ratios of DPPC and ApoA-I or ApoA-I mutants in rHDL assayThe weight ratios of DPPC to apoA-I in rHDL74 was slightly higher than in rHDLwt（1.93±0.06 vs.1.75±0.11）.However,this ratios in rHDL228（0.82±0.05 vs.1.75±0.11,P＜0.05）,rHDL107（1.49±0.12 vs.1.75±0.11,P＜0.05） and rHDL129 （1.47±0.04 vs.1.75±0.11,P＜0.05） were significantly lower than in rHDLwt.No significant alteration was observed in other rHDL groups.CONCLUSION:1.RHDL74 and rHDL52 are more effective than rHDLwt at reducing TNF-a and IL-1β.2.RHDL74 are more effective than rHDLwt at reducing IL-6.3.RHDL74 and rHDL52 are more effective than rHDLwt at attenuating endotoxin-induced clinical symptoms and protecting lung against LPS induced injury.4.Compared with rHDLwt,rHDL228 can significantly increase plasma concentration of TNF-αand exacerbate LPS induced lung injury.5.The weight ratios of DPPC to apoA-I in rHDL74 was slightly higher than in rHDLwt,However,this ratios in rHDL228 was the lowest among these rHDLs.In summary,for the first time,our study first compared the different effects of rHDL containing apoA-I cysteine mutants on the in vivo LPS induced endotoxemia in mice.Our data suggested that cysteine mutation not only induces the alteration of secondary structure and in vitro functions of apoA-I but also influences the capability of HDL to neutralize endotoxin or LPS.Due to its significant positive protection against LPS induced endotoxemia in our study,rHDL74 may be a potential clinical candidate for therapy of endotoxin induced septic shock.更多还原