白鲜皮抗内毒素的物质基础及其作用机制研究
摘要
一、背景
脓毒症(sepsis)是感染引起的全身性炎症反应综合征(systemic inflammatory response syndrome,SIRS),是严重烧伤、创伤、感染、休克和大手术等后常见的并发症,进一步发展可导致脓毒性休克、多器官系统功能障碍综合征(MODS)等,目前已成为临床危重患者的主要死亡原因之一。大量研究表明,革兰阴性(Gram-negative,G-)菌的内毒素(lipopolysaccharide,LPS)是介导脓毒症的主要启动子,LPS经通过活性中心类脂A(lipid A)与相应受体TLR4(Toll like 4,TLR4)结合,诱导炎症反应细胞合成与释放多种炎症介质(如TNF-α、IL-1和IL-6等),介导脓毒症的发生。尽管对脓毒症的病理生理及防治策略已进行较为深入的研究,但迄今为止临床上尚无特殊有效的治疗措施。由于LPS的胞内信号转导极为复杂,LPS诱发的细胞活化及细胞因子级联效应一旦启动将不易控制。因此,阻断LPS的活性中心lipid A与其受体的结合是防治脓毒症的关键。
中医中药是我国巨大的资源宝库,中医药在治疗脓毒症方面具有悠久历史。研究表明,多种中药具有抗菌、抗LPS作用,国内外研究也表明,来自天然的先导化合物中很有希望得到治疗疑难病症的新药,从天然化合物筛选得到的有效单体的命中率比合成化合物高。因此,如果能够从中药分离制备到与LPS作用的有效物质/单体,对开辟新的拮抗LPS措施具有重要意义。但由于中草药的化学组成多而复杂,其抗LPS作用的物质基础一直未得以阐明,其原因主要与中药单体研究中抗LPS靶点不明确及对活性组分无法及时跟踪检测有关。
因此,本研究采用生物传感器技术,将LPS的生物活性中心lipid A包被于生物传感器疏水样品池,使lipid A结构上发挥重要生物学作用的亲水端外露,以此作为中药抗LPS活性组分筛选、跟踪和检测的靶点,从60种中药中选择出与lipid A亲和力较高的中药白鲜皮作为研究对象,结合传统的中药分离技术及高效液相色谱(high performance liquid chromatogram,HPLC)等方法,从白鲜皮水煎液中获得抗LPS的活性物质进行体外内抗LPS的活性研究。
二、目的
从中药白鲜皮中分离提取具有拮抗LPS活性的物质并研究其作用机制。
三、材料与方法
1.应用lipid A为靶点的生物传感器技术平台,观察60种中药水煎液与lipid A的结合反应;以白鲜皮水煎液为研究对象,通过大孔吸附树脂、正丁醇萃取、聚酰胺柱层析和HPLC,分离提取其中与lipid A的高亲和力结合的组分或产物;动态浊度法鲎试验检测HPLC分离产物DPR1~4对LPS的中和活性;通过质谱、红外光谱和核磁共振谱对其中的活性物质DPR-2进行结构分析。
2.应用生物传感器技术平台,观察DPR-2与lipid A的结合作用并计算二者的平衡解离常数(Kd)值;动态浊度法鲎试验测定DPR-2对LPS的中和能力;激光扫描共聚焦显微镜(LSM)观察不同浓度DPR-2(0、8、16、32、64μg/ml)下异硫氰酸荧光素标记LPS(FITC-LPS,100 ng/ml)与小鼠单核/巨噬细胞RAW264.7细胞结合的荧光强度;免疫细胞化学法观察DPR-2干预后LPS(100 ng/ml)诱导的RAW264.7细胞TLR4的表达;采用实时荧光定量反转录聚合酶链反应(real-time RT-PCR)和ELISA法检测DPR-2干预后LPS(100 ng/ml)刺激的RAW264.7细胞TLR4、TNF-α和IL-6 mRNA的表达及细胞因子TNF-α和IL-6的分泌;MTT法检测DPR-2对RAW264.7细胞活力的影响。
3.采用尾静脉注射LPS制备小鼠脓毒症和内毒素血症(endotoxemia,ETM)模型;观察DPR-2对致死剂量LPS攻击小鼠的保护作用;观察DPR-2对ETM小鼠的治疗作用:伤后2、6、12、24、48和72 h采集血和肝、肺组织标本,动态浊度法鲎试验检测血浆LPS水平;ELISA法检测血浆TNF-α、IL-6水平;Real-time RT-PCR检测肝、肺组织的TNF-α和IL-6 mRNA表达;常规HE染色观察肝、肺的病理学变化。
四、结果
1.大黄、青果、赤芍、白鲜皮等4种中药水煎液与lipid A具有较高的特异性结合能力(RU>300 arc seconds)。从白鲜皮中提取活性物质DPR-2,结构分析可能是一种分子量为709的四糖化合物。
2. DPR-2与lipid A具有较高的亲和力,其Kd值为8.63×10-6M,DPR-2体外对LPS具有中和作用。DPR-2可抑制FITC-LPS与小鼠单核/巨噬细胞RAW264.7细胞膜受体结合,并对LPS诱导的RAW264.7细胞TLR4、TNF-α及IL-6在蛋白及基因表达水平均具有抑制作用并呈剂量效应关系。DPR-2在体外实验浓度对细胞活力无影响。
3. DPR-2对脓毒症模型小鼠具有显著的保护作用,可降低ETM小鼠的血浆LPS、TNF-α及IL-6水平,减弱肝、肺组织的TNF-α及IL-6 mRNA表达,并减轻ETM小鼠重要器官肝、肺的病理学损伤。
五、结论
本论文采用生物传感器技术,以LPS的生物活性中心lipid A作为筛选、跟踪中药活性组分或物质的靶点,结合传统的中药分离技术及HPLC等方法,从筛选出的中药白鲜皮中分离获得高亲和力结合lipid A的活性物质DPR-2(分子量为709的四糖化合物),研究表明DPR-2对LPS攻击小鼠具有保护作用,其机制可能与DPR-2的拮抗LPS活性有关,而DPR-2与lipid A的高亲和力结合活性可能是其体内外拮抗LPS活性的重要基础。
Introduction Sepsis is a complex clinical syndrome that results from a harmful host response to infection, which may result in septic shock, multiple organ failure (MOF) and ultimately death. A recent epidemiological study from North America found that the incidence was approximately 3.0 cases per 1,000 population, which translated into an annual burden of approximately 750,000 cases. The overall mortality is approximately 30%, rising to 40% in the elderly and is 50% or greater in patients with the most severe syndrome, septic shock. The most common cause of sepsis is an exposure to the structural component of the Gram-negative bacterial outer membrane, lipopolysaccharide (LPS, known also as endotoxin).
LPS is thought to be the main toxic element that induces pro-inflammatory cytokine production after interaction with CD14 and toll-like receptor 4 (TLR4). Pro-inflammatory cytokines such as TNF-α, IL-1 and IL-6 are induced by LPS, which may damage cells and lead to organ injuries. Despite improved care, the hospital mortality rate from severe sepsis and septic shock has not improved significantly over recent decades. Thus, it is important to investigate new anti-LPS drugs to identify a relevant anti-sepsis drug.
The use of traditional Chinese medicines has a long history in sepsis therapy. A lot of herbs have been shown to possess potent anti-LPS and anti-inflammatory properties. However, herbs possess a large number of complex constituents and it is extremely difficult to identify which is the anti-LPS component. In the present study, we undertook to confirm the presence of anti-LPS component(s) in a panel of 60 traditional Chinese herbs, using affinity biosensor technology. The herb, densefruit pittany root-bark, was selected from this panel, and components were then isolated that possessed the highest lipid A-binding ability. Finally, the active compound isolated from densefruit pittany root-bark was examined for its anti-LPS activity both in vitro and in vivo.
Objective To obtain the active compound from traditional Chinese herbs that can antagonize LPS and investigate its associated mechanism.
Methods 1. Lipid A was immobilized on the optical biosensor's hydrophobic cuvette and employed to monitor the interactions of the components from Chinese herbs with lipid A. Sixty aqueous extractions of traditional Chinese medicine were determined the exact capacity for binding to lipid A using the affinity biosensor technology. The aqueous extraction from densefruit pittany root-bark, which possessed higher binding activity to lipid A, were selected for further study. Briefly, the fraction obtained was subjected to macroporous adsorptive resins, n-butanol solvent extraction, polyamide laminar analysis, and the fraction with the highest binding activity to lipid A was further purified by high performance liquid chromatography (HPLC). Then the neutralization of HPLC-purified products DPR1~4 for LPS (0.1 ng/ml) was detected by kinetic turbidimetric limulus test. 2. The Kd value for the lipid A of active compound, DPR-2, was measured by biosensor, and the activity of DPR-2 (0.5, 1, 2 and 4μg/ml) neutralizing LPS (0.1 ng/ml) was detected by kinetic turbidimetric limulus test. Then the binding of FITC-conjugated LPS (100 ng/ml) to murine RAW264.7 cells was analyzed by laser scanning confocal microscopy (LSM) and the influence of DPR-2 (8, 16, 32 and 64μg/ml) on TLR4 expression in RAW264.7 cells simulated with LPS (100ng/ml) was detected by immunocytochemical staining. Meanwhile, the expressions of TLR4, TNF-αand IL-6 mRNA and the release of cytokines were detected with real-time RT-PCR and ELISA, respectively. 3. In vivo, different dosage of LPS (2.5, 5, 10, 15 and 20mg/kg body wt) was injected into BALB/c mice by tail vein to prepare the murine model of sepsis and endotoxemia murine model, and the effects of DPR-2 were observed. Then, in an endotoxemia murine model (12mg/kg body wt), blood samples were collected at different time (2, 6, 12, 24, 48 and 72 h) after inception of the experiment and assayed using the appropriate ELISA kits, and the TNF-αand IL-6 mRNA expressions in liver and lung were analysed by real-time RT-PCR. Meanwhile, pathological changes of hepatic tissue and lung tissue were observed.
Results 1. Among the 60 Chinese herbs examined, four herbs were found to possess higher lipid A-binding activities (RU>300 arc seconds). Four chemical components DPR1~4 with different affinity for lipid A from densefruit pittany root-bark were obtained by above methods. The active compound DPR-2 was purified and confirmed as a compound with tetrasaccharide. 2. DPR-2 bound with high-affinity to lipid A with Kd value of 8.63μM and neutralized LPS in a dose-dependent manner. Furthermore, DPR-2 inhibited the binding of FITC-LPS to RAW264.7 cells in a dose-dependent manner and TLR4 expression of RAW264.7 cells induced by LPS. Moreover, DPR-2 could inhibit markedly the expressions of TNF-αand IL-6 mRNA and the release of cytokines in LPS-stimulated murine RAW264.7 cells. 3. DPR-2 was found to protect BALB/c mice from lethal challenge by LPS and decreased the plasma LPS level in endotoxemic mice. In the endotoxemia murine model, DPR-2 could potently suppress LPS-induced expression of TNF-αand IL-6 mRNA in hepatic tissue and lung tissue and significantly attenuate the release of cytokines in plasma. In addition, it could alleviate the pathological changes in liver and lung.
Conclusion The active compound, DPR-2, was obtained from aqueous extraction of densefruit pittany root-bark using biosensor technology. DPR-2 was found to neutralize LPS and possess anti-LPS capacity in vitro and in vivo. This might be associated with its hight-affinity for lipid A.
脓毒症(sepsis)是感染引起的全身性炎症反应综合征(systemic inflammatory response syndrome,SIRS),是严重烧伤、创伤、感染、休克和大手术等后常见的并发症,进一步发展可导致脓毒性休克、多器官系统功能障碍综合征(MODS)等,目前已成为临床危重患者的主要死亡原因之一。大量研究表明,革兰阴性(Gram-negative,G-)菌的内毒素(lipopolysaccharide,LPS)是介导脓毒症的主要启动子,LPS经通过活性中心类脂A(lipid A)与相应受体TLR4(Toll like 4,TLR4)结合,诱导炎症反应细胞合成与释放多种炎症介质(如TNF-α、IL-1和IL-6等),介导脓毒症的发生。尽管对脓毒症的病理生理及防治策略已进行较为深入的研究,但迄今为止临床上尚无特殊有效的治疗措施。由于LPS的胞内信号转导极为复杂,LPS诱发的细胞活化及细胞因子级联效应一旦启动将不易控制。因此,阻断LPS的活性中心lipid A与其受体的结合是防治脓毒症的关键。
中医中药是我国巨大的资源宝库,中医药在治疗脓毒症方面具有悠久历史。研究表明,多种中药具有抗菌、抗LPS作用,国内外研究也表明,来自天然的先导化合物中很有希望得到治疗疑难病症的新药,从天然化合物筛选得到的有效单体的命中率比合成化合物高。因此,如果能够从中药分离制备到与LPS作用的有效物质/单体,对开辟新的拮抗LPS措施具有重要意义。但由于中草药的化学组成多而复杂,其抗LPS作用的物质基础一直未得以阐明,其原因主要与中药单体研究中抗LPS靶点不明确及对活性组分无法及时跟踪检测有关。
因此,本研究采用生物传感器技术,将LPS的生物活性中心lipid A包被于生物传感器疏水样品池,使lipid A结构上发挥重要生物学作用的亲水端外露,以此作为中药抗LPS活性组分筛选、跟踪和检测的靶点,从60种中药中选择出与lipid A亲和力较高的中药白鲜皮作为研究对象,结合传统的中药分离技术及高效液相色谱(high performance liquid chromatogram,HPLC)等方法,从白鲜皮水煎液中获得抗LPS的活性物质进行体外内抗LPS的活性研究。
二、目的
从中药白鲜皮中分离提取具有拮抗LPS活性的物质并研究其作用机制。
三、材料与方法
1.应用lipid A为靶点的生物传感器技术平台,观察60种中药水煎液与lipid A的结合反应;以白鲜皮水煎液为研究对象,通过大孔吸附树脂、正丁醇萃取、聚酰胺柱层析和HPLC,分离提取其中与lipid A的高亲和力结合的组分或产物;动态浊度法鲎试验检测HPLC分离产物DPR1~4对LPS的中和活性;通过质谱、红外光谱和核磁共振谱对其中的活性物质DPR-2进行结构分析。
2.应用生物传感器技术平台,观察DPR-2与lipid A的结合作用并计算二者的平衡解离常数(Kd)值;动态浊度法鲎试验测定DPR-2对LPS的中和能力;激光扫描共聚焦显微镜(LSM)观察不同浓度DPR-2(0、8、16、32、64μg/ml)下异硫氰酸荧光素标记LPS(FITC-LPS,100 ng/ml)与小鼠单核/巨噬细胞RAW264.7细胞结合的荧光强度;免疫细胞化学法观察DPR-2干预后LPS(100 ng/ml)诱导的RAW264.7细胞TLR4的表达;采用实时荧光定量反转录聚合酶链反应(real-time RT-PCR)和ELISA法检测DPR-2干预后LPS(100 ng/ml)刺激的RAW264.7细胞TLR4、TNF-α和IL-6 mRNA的表达及细胞因子TNF-α和IL-6的分泌;MTT法检测DPR-2对RAW264.7细胞活力的影响。
3.采用尾静脉注射LPS制备小鼠脓毒症和内毒素血症(endotoxemia,ETM)模型;观察DPR-2对致死剂量LPS攻击小鼠的保护作用;观察DPR-2对ETM小鼠的治疗作用:伤后2、6、12、24、48和72 h采集血和肝、肺组织标本,动态浊度法鲎试验检测血浆LPS水平;ELISA法检测血浆TNF-α、IL-6水平;Real-time RT-PCR检测肝、肺组织的TNF-α和IL-6 mRNA表达;常规HE染色观察肝、肺的病理学变化。
四、结果
1.大黄、青果、赤芍、白鲜皮等4种中药水煎液与lipid A具有较高的特异性结合能力(RU>300 arc seconds)。从白鲜皮中提取活性物质DPR-2,结构分析可能是一种分子量为709的四糖化合物。
2. DPR-2与lipid A具有较高的亲和力,其Kd值为8.63×10-6M,DPR-2体外对LPS具有中和作用。DPR-2可抑制FITC-LPS与小鼠单核/巨噬细胞RAW264.7细胞膜受体结合,并对LPS诱导的RAW264.7细胞TLR4、TNF-α及IL-6在蛋白及基因表达水平均具有抑制作用并呈剂量效应关系。DPR-2在体外实验浓度对细胞活力无影响。
3. DPR-2对脓毒症模型小鼠具有显著的保护作用,可降低ETM小鼠的血浆LPS、TNF-α及IL-6水平,减弱肝、肺组织的TNF-α及IL-6 mRNA表达,并减轻ETM小鼠重要器官肝、肺的病理学损伤。
五、结论
本论文采用生物传感器技术,以LPS的生物活性中心lipid A作为筛选、跟踪中药活性组分或物质的靶点,结合传统的中药分离技术及HPLC等方法,从筛选出的中药白鲜皮中分离获得高亲和力结合lipid A的活性物质DPR-2(分子量为709的四糖化合物),研究表明DPR-2对LPS攻击小鼠具有保护作用,其机制可能与DPR-2的拮抗LPS活性有关,而DPR-2与lipid A的高亲和力结合活性可能是其体内外拮抗LPS活性的重要基础。
Introduction Sepsis is a complex clinical syndrome that results from a harmful host response to infection, which may result in septic shock, multiple organ failure (MOF) and ultimately death. A recent epidemiological study from North America found that the incidence was approximately 3.0 cases per 1,000 population, which translated into an annual burden of approximately 750,000 cases. The overall mortality is approximately 30%, rising to 40% in the elderly and is 50% or greater in patients with the most severe syndrome, septic shock. The most common cause of sepsis is an exposure to the structural component of the Gram-negative bacterial outer membrane, lipopolysaccharide (LPS, known also as endotoxin).
LPS is thought to be the main toxic element that induces pro-inflammatory cytokine production after interaction with CD14 and toll-like receptor 4 (TLR4). Pro-inflammatory cytokines such as TNF-α, IL-1 and IL-6 are induced by LPS, which may damage cells and lead to organ injuries. Despite improved care, the hospital mortality rate from severe sepsis and septic shock has not improved significantly over recent decades. Thus, it is important to investigate new anti-LPS drugs to identify a relevant anti-sepsis drug.
The use of traditional Chinese medicines has a long history in sepsis therapy. A lot of herbs have been shown to possess potent anti-LPS and anti-inflammatory properties. However, herbs possess a large number of complex constituents and it is extremely difficult to identify which is the anti-LPS component. In the present study, we undertook to confirm the presence of anti-LPS component(s) in a panel of 60 traditional Chinese herbs, using affinity biosensor technology. The herb, densefruit pittany root-bark, was selected from this panel, and components were then isolated that possessed the highest lipid A-binding ability. Finally, the active compound isolated from densefruit pittany root-bark was examined for its anti-LPS activity both in vitro and in vivo.
Objective To obtain the active compound from traditional Chinese herbs that can antagonize LPS and investigate its associated mechanism.
Methods 1. Lipid A was immobilized on the optical biosensor's hydrophobic cuvette and employed to monitor the interactions of the components from Chinese herbs with lipid A. Sixty aqueous extractions of traditional Chinese medicine were determined the exact capacity for binding to lipid A using the affinity biosensor technology. The aqueous extraction from densefruit pittany root-bark, which possessed higher binding activity to lipid A, were selected for further study. Briefly, the fraction obtained was subjected to macroporous adsorptive resins, n-butanol solvent extraction, polyamide laminar analysis, and the fraction with the highest binding activity to lipid A was further purified by high performance liquid chromatography (HPLC). Then the neutralization of HPLC-purified products DPR1~4 for LPS (0.1 ng/ml) was detected by kinetic turbidimetric limulus test. 2. The Kd value for the lipid A of active compound, DPR-2, was measured by biosensor, and the activity of DPR-2 (0.5, 1, 2 and 4μg/ml) neutralizing LPS (0.1 ng/ml) was detected by kinetic turbidimetric limulus test. Then the binding of FITC-conjugated LPS (100 ng/ml) to murine RAW264.7 cells was analyzed by laser scanning confocal microscopy (LSM) and the influence of DPR-2 (8, 16, 32 and 64μg/ml) on TLR4 expression in RAW264.7 cells simulated with LPS (100ng/ml) was detected by immunocytochemical staining. Meanwhile, the expressions of TLR4, TNF-αand IL-6 mRNA and the release of cytokines were detected with real-time RT-PCR and ELISA, respectively. 3. In vivo, different dosage of LPS (2.5, 5, 10, 15 and 20mg/kg body wt) was injected into BALB/c mice by tail vein to prepare the murine model of sepsis and endotoxemia murine model, and the effects of DPR-2 were observed. Then, in an endotoxemia murine model (12mg/kg body wt), blood samples were collected at different time (2, 6, 12, 24, 48 and 72 h) after inception of the experiment and assayed using the appropriate ELISA kits, and the TNF-αand IL-6 mRNA expressions in liver and lung were analysed by real-time RT-PCR. Meanwhile, pathological changes of hepatic tissue and lung tissue were observed.
Results 1. Among the 60 Chinese herbs examined, four herbs were found to possess higher lipid A-binding activities (RU>300 arc seconds). Four chemical components DPR1~4 with different affinity for lipid A from densefruit pittany root-bark were obtained by above methods. The active compound DPR-2 was purified and confirmed as a compound with tetrasaccharide. 2. DPR-2 bound with high-affinity to lipid A with Kd value of 8.63μM and neutralized LPS in a dose-dependent manner. Furthermore, DPR-2 inhibited the binding of FITC-LPS to RAW264.7 cells in a dose-dependent manner and TLR4 expression of RAW264.7 cells induced by LPS. Moreover, DPR-2 could inhibit markedly the expressions of TNF-αand IL-6 mRNA and the release of cytokines in LPS-stimulated murine RAW264.7 cells. 3. DPR-2 was found to protect BALB/c mice from lethal challenge by LPS and decreased the plasma LPS level in endotoxemic mice. In the endotoxemia murine model, DPR-2 could potently suppress LPS-induced expression of TNF-αand IL-6 mRNA in hepatic tissue and lung tissue and significantly attenuate the release of cytokines in plasma. In addition, it could alleviate the pathological changes in liver and lung.
Conclusion The active compound, DPR-2, was obtained from aqueous extraction of densefruit pittany root-bark using biosensor technology. DPR-2 was found to neutralize LPS and possess anti-LPS capacity in vitro and in vivo. This might be associated with its hight-affinity for lipid A.
引文
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1. Zahorec R, Firment J, Strakova J, Mikula J, Malik P, Novak I, Zeman J, Chlebo P. Epidemiology of severe sepsis in intensive care units in the Slovak Republic. Infection, 2005, 33(3): 122-128.
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4. Levy MM, Fink MP, Marshall JC, Abraham E, Angus D, Cook D, Cohen J, Opal SM, Vincent JL, Ramsay G. 2001 SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference. Crit-Care-Med, 2003, 31(4): 1250-1256.
5. Takeuchi O, Sato S, Horiuchi T, Hoshino K, Takeda K, Dong Z, Modlin RL, Akira S. Cutting edge: role of Toll-like receptor 1 in mediating immune response to microbial lipoproteins. J-Immunol, 2002, 169(1): 10-14.
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7. Asai Y, Ohyama Y, Gen K, Ogawa T. Bacterial fimbriae and their peptides activate human gingival epithelial cells through Toll-like receptor 2. Infect-Immun, 2001, 69(12): 7387-7395.
8. Alexopoulou L, Holt AC, Medzhitov R, Flavell RA. Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature, 2001, 413(6857): 732-738.
9. Hoshino K, Takeuchi O, Kawai T, Sanjo H, Ogawa T, Takeda Y, Takeda K, Akira S. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J-Immunol, 1999, 162(7): 3749-3752.
10. Netea MG, van-Deuren M, Kullberg BJ, Cavaillon JM, Van-der-Meer JW. Does the shape of lipid A determine the interaction of LPS with Toll-like receptors?Trends-Immunol, 2002, 23(3): 135-139.
11. Ohashi K, Burkart V, Flohe S, Kolb H. Cutting edge: heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex. J-Immunol, 2000, 164(2): 558-561.
12. Gao B, Tsan MF. Endotoxin contamination in recombinant human heat shock protein
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