PEG化重组人白细胞介素-6制品游离PEG测定方法的研究

为了建立PEG化学修饰的重组人白细胞介素-6(PEG-rhIL-6)的部分质量控制方法,参照2005年版《中华人民共和国药典》三部附录III B高效液相色谱法,以RP-HPLC方法分离PEG-rhIL-6原液中的不同成分,用蒸发光散射监测器检测游离PEG,用外标法测定计算样品中残留PEG的含量。PEG修饰rhIL-6结构稳定;制品中的游离PEG含量符合要求。RP-HPLC法检测游离PEG结果可靠。     

大肠杆菌表达的人IL-6复性条件研究

采用稀释法复性,筛选重组人白细胞介素-6(rhIL-6)包涵体蛋白的复性条件。结果显示,复性液的浓度、pH值、复性时间和复性蛋白浓度,对重组人白细胞介素-6复性效果有很大影响。在复性液为2mol/L尿素、pH8.5、复性时间为24h和复性蛋白浓度50μg/ml条件下,重组人白细胞介素-6包涵体蛋白的复性效果最佳。     

血小板减小症模型制作及药效试验应用研究

[目的] 研究血小扳减少症模型复制的方法和皮下注射白细胞介索-6对小鼠血小板减少症治疗的效果。[方法] 采用12只BACB/C小鼠,雌雄各半,随机分为3组,皮下注射环磷酰胺;再将3个模型组随机设为阴性对照组、阳性对照组以及白细胞介索-6实验组,分别皮下注射稀释液、白细胞介素-11及白细胞介素-6,定时采血,分析血小板数目。[结果] ①与给药前相比,小鼠血小板减少极显著(P＜0．01);②阴性对照组与阳性对照组以及白细胞介素-6实验组之间差异极显著(P＜0．01）;③阳性对照组、白细胞介素-6实验组给药前相比差异极显著(P＜0．01)。[结论] 用环磷酰胺对BACB/C小鼠注射方法制造模型简单易行;白细胞介素-6在BACB/C小鼠以皮下注射给药途径方法,对治疗小鼠血小板减少症效果显著。     

猪白细胞介素2与融合白细胞介素4/6基因共表达的免疫效应研究

目的构建猪融合白细胞介素4/6与猪白细胞介素2基因的共表达载体,研究其共表达对小鼠免疫的协同效应。方法以2A自剪接技术首次构建猪融合白细胞介素4/6与白细胞介素2基因的共表达重组质粒,以壳聚糖纳米材料包裹制成纳米颗粒,进行体外转染HEK293细胞,提取总RNA进行RT-PCR分析,最后肌肉注射小鼠进行体内实验并分析。结果成功构建了重组共表达质粒VRIL4/6-2;壳聚糖包裹后转染HEK293细胞,48 h后收集细胞,RT-PCR检测显示目的基因能够在HEK293细胞中高效地转录与表达。小鼠接种实验结果表明,实验组血清中的IgG和IgG2a的含量比对照组显著增加,并且VRIL4/6-2-CS接种组的小鼠体液免疫水平明显增高(P0.05);VRIL4/6-2实验组的CD4+淋巴细胞显著多于其它实验组(P0.05);实验组IL-2、IL-4、IL-6、IL-23基因的表达水平明显高于对照组(P0.05),实验组小鼠外周血白细胞、血小板和血红蛋白的含量均显著高于对照组(P0.05)。结论 VRIL4/6-2共表达质粒能够更好的增强动物体液免疫和细胞免疫机能,可作为提高动物体液免疫和细胞免疫的经济高效安全新型免疫调节剂,具有潜在的重要应用前景。     

rhIL 1β、rhIL 2和rhIL 6诱导体外培养大鼠海马神经元Fos表达——免疫组织化学研究

采用免疫组织化学方法, 观察了人重组白细胞介素１ （ｒｈＩＬ１β）、人重组白细胞介素２ （ｒｈＩＬ２）和人重组白细胞介素６ （ｒｈＩＬ６） 诱导体外培养大鼠海马神经元Ｆｏｓ表达。结果显示, 培养１２ ｄ 的海马神经元分别与ｒｈＩＬ１β、ｒｈＩＬ２ 和ｒｈＩＬ６ 培养２ｈ 后, Ｆｏｓ免疫反应（ＦｏｓＩＲ） 阳性细胞百分率增多。随着所给剂量增加,ＦｏｓＩＲ阳性细胞百分率明显增多。图像分析的结果显示,ＦｏｓＩＲ阳性细胞的平均光密度亦随所给剂量的增大而增加。以上结果表明, ｒｈＩＬ１β、ｒｈＩＬ２ 和ｒｈＩＬ６ 均能诱导体外培养大鼠海马神经元Ｆｏｓ表达。提示ｒｈＩＬ１β、ｒｈＩＬ２ 和ｒｈＩＬ６ 对体外培养的大鼠海马神经元具有生物学作用。推测Ｆｏｓ表达可能是ｒｈＩＬ１β、ｒｈＩＬ２ 和ｒｈＩＬ６ 等细胞因子发挥生物效应的重要中间途径。     

重组人白细胞介素-6对缺氧-复氧后大鼠海马培养神经元Bcl-2、Bax 表达和神经元凋亡的影响

实验在培养的大鼠海马神经元中观察了重组人白细胞介素-6（recombinant human interleukin-6,rhIL-6）对缺氧-复氧后Bcl-2、Bax表达和神经元凋亡的影响。把培养12d的大鼠海马神经元分为对照组和rhIL-6组,同时于缺氧环境（90% N2 10% CO2）中培养2、4h后,再于常氧培养箱内复氧培养24和72h。于不同时间取出,分别用抗Bcl-2和Bax抗血清进行免疫组织化学染色,观察缺氧-复氧后大鼠海马培养神经元Bcl-2和Bax的表达,并用原位末端标记（TUNEL）法和流式细胞术分别检测缺氧-复氧对体外培养海马神经元凋亡的影响。结果可见,与缺氧前相比,缺氧-复氧后24和72h,海马神经元Bal-2表达明显减弱,Bax表达明显增强,凋亡神经元明显增多。经rhIL-6预处理的海马神经元与对照组相比,缺氧-复氧后24和72h,Bcl-2表达明显增强,Bax神经明显减弱,凋亡神经元明显减少。本实验结果提示,rhIL-6对海马神经元缺氧-复氧损伤具有一定的保护作用。     

聚乙二醇化重组人白细胞介素-6经不同途径给药的药代动力学比较研究

目的 研究聚乙二醇化重组人白细胞介素-6不同给药途径的药代动力学.方法 将大鼠分为皮下给药组、静脉给药组,每组各设3个剂量组.分别按40μg/kg、20μg/kg、3μg/kg给药.同位素示踪法用碘标记PEGrhIL-6,采用TCA沉淀法检测放射性浓度,3P87软件判断房室模型并计算各种参数,并检测125I-PEG-rhIL-6皮下注射大鼠后不同时间的血药浓度.结果 ①静脉给药大鼠体内的血药浓度-时间曲线符合二房室模型,而皮下给药大鼠体内的血药浓度-时间曲线符合一房室模型;②皮下给药的达峰时间比静脉给药慢,但其有效血药浓度维持时间较静脉给药长;③皮下给药较静脉给药各时点血药浓度低.结论 皮下给药毒性低,是一种安全可靠的给药方法;同时有效血药浓度维持时间较长,有利于治疗血小板减少症.     

聚乙二醇化重组人粒细胞集落刺激因子的药效学研究

比较研究聚乙二醇化重组人粒细胞集落刺激因子(PEG-rhG-CSF)与重组人粒细胞集落刺激因子(rhG-CSF)升高环磷酰胺所致外周血白细胞降低的Blab/c小鼠的白细胞效果.结果显示一次注射聚乙二醇化重组人粒细胞集落刺激因子与多次注射重组人粒细胞集落刺激因子的药效作用相当,且各剂量间呈良好的量效关系.     

骨髓基质细胞IL-1beta、IL-6、SCF及TPO在老年多发性骨髓瘤组织中 的表达及意义

目的:探讨老年多发性骨髓瘤患者骨髓基质细胞白细胞介素-1(IL-1β)、白细胞介素-6(IL-6)、肝细胞因子(SCF)、血小板生成素(TPO)水平及临床意义。方法:选择本院2011年1月-2014年6月收治的老年多发性骨髓瘤患者作为观察组,另选择同期参加体检的志愿者作为对照组。采集两组人员骨髓标本,制备总RNA、反转录反应、聚合酶链反应及PCR产物电泳检测等过程的测定两组患者IL-1β、IL-6、SCF、TPO水平。结果:观察组患者IL-1β、IL-6、SCF、TPO水平均明显高于对照组(P<0.05);初发患者、复发患者及移植患者的IL-1β、IL-6比较无明显差异(P>0.05)。移植组SCF水平显著低于初发组和复发组(t初移=4.967,t难移=5.169,P<0.05);初发组TPO显著高于复发组和移植组(t初难=4.736,t初移=3.568,P<0.05)。结论:老年多发性骨髓瘤患者骨髓基质细胞白细胞介素-1、白细胞介素-6、肝细胞因子、血小板生成素水平表达升高,在疾病的发生及发展过程中发挥重要作用。     

维生素D及白细胞介素-6水平在 结直肠癌患者预后评估中的价值

目的探讨维生素D及白细胞介素-6水平在结直肠癌患者预后评估中的价值。方法选取树兰(杭州)医院2016-2017年经病理学证实的结直肠癌患者45例和同期健康体检者53例,检测患者外周血中维生素D、白细胞介素-6水平。根据检测结果,将结直肠癌患者分为维生素D高表达组和低表达组以及白细胞介素-6高表达组和低表达组,分别比较各组患者临床病理参数,并对患者进行预后评估。结果结直肠癌患者外周血中维生素D含量为(6.43±3.71)ng/mL,低于健康对照组的(10.21±3.54)ng/mL,(P0.01);白细胞介素-6水平为17.5(8.97~42.92)ng/mL,高于健康对照组的9.15(3.51~13.79)ng/mL,(P0.01),且二者在结直肠癌组织中的水平可能具有相关性(χ2=7.4,P0.01)。维生素D及白细胞介素-6水平与结直肠癌患者是否发生淋巴结核转移、TNM分期和肿瘤浸润深度密切相关。预后分析发现,血清维生素D浓度与患者预后呈正相关,血清白细胞介素-6水平与患者预后呈负相关。结论维生素D及白细胞介素-6水平与结直肠癌患者预后有关,可作为判断结直肠癌患者预后的新型、有效的生物学指标。     

抗人IL-6单克隆抗体的制备及鉴定

用基因重组人IL-6免疫Balb/c小鼠,采用小鼠杂交瘤技术,筛选克隆到分泌抗人重组IL-6单克隆抗体的杂交瘤细胞株,并对其中2H₂、 1D₂ 和4B₄瘤细胞株进行了鉴定.其抗体类别均为IgG,亚类分别为IgG1和IgG2a.用多种细胞因子和无关蛋白的鉴别试验结果证实它们均特异地识别rhIL-6.免疫转染结果显示,该单抗识别分子质量为21 ku的IL-6单一条带.IL-6单克隆抗体的亲和常数K_aff= 1.62×10⁹ (mol/L)^-1.     

Interleukin 6 knock-out mice are resistant to antigen-induced experimental arthritis

In order to assess the potential role of IL-6 in rheumatoid arthritis (RA), we have compared IL-6 deficient (IL-6 ko) mice and their wild-type (wt) counterpart for the capacity to develop methylated bovine serum albumin (mBSA)-induced arthritis. Our data show that IL-6 ko mice are not susceptible to antigen-induced arthritis (AIA). In fact, IL-6 ko mice treated by a standard protocol of immunization with mBSA did not develop joint swelling following intra-articular mBSA injection, nor revealed the characteristic joint lesions by histological examination. Conversely, wt mice treated according to the same protocol developed arthritis about 9 days after intra-articular injection, as detected by knee joint swelling and histological confirmation. We observed that the proliferative response of splenocytes to mBSA was impaired in ko mice following arthritis induction, as compared to the strong response observed in wt mice. Furthermore, anti-mBSA IgG levels were lower in ko mice as compared to wt mice. Finally, we show that sensitivity to AIA can be reconstituted in ko mice by subcutaneous injections of recombinant human IL-6 (rhIL-6). In addition, co-administration of IL-6 with mBSA by intra-articular injection into the joint was only partially effective in conferring sensitivity to AIA, suggesting the importance of a systemic effct for IL-6, but also that an additional role for this cytokine can be envisaged in the local inflammatory reaction during establishment of AIA.     

Interleukin-6 is antiproliferative to a mouse hybridoma cell line and promotive for its antibody productivity

Monoclonal antibody production by hybridoma cells at moderately slowed growth states would be favorable for commercial scale production since cells can devote their resources to performing the differentiated function, immunoglobulin production. We found that a purified recombinant human interleukin-6, which had been reported to support or stimulate proliferation of B cell hybridoma/plasmacytoma cells, suppressed growth of a hybridoma cell line in serum-free medium. In the presence of the interleukin, the growth-suppressed cells were viable for remarkably long periods in batch culture, and after removal of the interleukin from the culture medium, they started to proliferate at their normal growth rate. As the concentration of the interleukin increased in the culture, the growth rate decreased and the specific antibody productivity (antibody production rate per cell) increased to 5-fold of control at 10 U ml^–1 (2 ng ml^–1) of the interleukin.Abbreviations IL-1,2, and 6 interleukin-1, 2 and 6 - rhIL-6 recombinant human interleukin-6 - MCAb monoclonal antibody - TNP trinitrophenyl - unit (U) of interleukin-6 A unit (U) is equivalent to the amounts of IL-6 which gives one-half maximal IgM secretion by SKW6-CL4 cells (1U ml^–1=200 pg ml^–1)     

Thrombopoietic activity of human interleukin-6

Thrombopoietin (TPO), a regulatory factor in platelet production, was purified from the conditioned medium of TNK-01 cells cultured in the presence of human interleukin-1. The N-terminal sequence of purified TPO was determined to be VPPGEDSKDVAAPHRQPLT, identical to that of the N-terminal region of human interleukin-6 (IL-6). Two forms of TPO with molecular masses of 24 and 27 kDa were identified as IL-6 by Western analysis using an anti-IL-6 antibody. Commercial recombinant human IL-6 produced in Escherichia coli, stimulated megakaryocyte colony formation in the presence of mouse interleukin-3 and increased the number of peripheral platelets in mice in a dose-dependent manner. From these results, it is concluded that human IL-6 has thrombopoietic activity.     

Plasma clearance, organ distribution and target cells of interleukin-6/hepatocyte-stimulating factor in the rat

The plasma half-life of recombinant human interleukin-6 (rhIL-6) was determined in rats by measuring the disappearance of the biological activity as well as of the radioactivity of 125I-rhIL-6 from the circulation. The kinetics of clearance were biphasic. It consisted of a rapid initial disappearance corresponding to a half-life of 3 min, and of a second slow one corresponding to a half-life of about 55 min. By cellulose-acetate electrophoresis it was shown that rhIL-6 binds to a plasma protein resulting in a complex migrating in the beta-gamma region; 20 min after intravenous injection, about 80% of the 125I-rhIL-6 that had disappeared from the circulation was found in the liver. 125I-rhIL-6 was exclusively localized on the surface of parenchymal cells suggesting the existence of an interleukin-6 receptor on the hepatocytes.     

Effect of recombinant human interleukin-6 on nitrite production of mouse myeloid leukemia cells

The effect of recombinant human interleukin-6 (rhIL-6) on induction of nitrite (NO(-2)) production was investigated in a mouse myeloid leukemia cell line (M1) and a subclone (Mm1). NO(-2) was induced by rhIL-6 (greater than 50 U/ml) in these cell lines. Pretreatment with rhIL-6 (100 U/ml) for 1 day synergistically enhanced the production of NO(-2) in Mm1 by LPS. Furthermore, pretreatment with IL-6 (100 U/ml) shortened the lag time of induction. These results indicate that IL-6 is involved in the regulation of the NO(-2) production in macrophage-like cells.     

Anti-interleukin 6 (IL-6) receptor antibody suppresses Castleman's disease like symptoms emerged in IL-6 transgenic mice

Transgenic mice carrying human IL-6 cDNA fused with a murine major histocompatibility class-I promoter (H-2L(d)) were serially administered with anti-interleukin-6 receptor (IL-6R) monoclonal antibody (mAb), MR16-1, from the age of 4 weeks to estimate its efficacy on a variety of disorders developed in these mice, most of which are similar to the disorders associated with Castleman's disease. In the control mice treated with isotype-matched mAb, a massive and multiple IgG1 plasmacytosis, mesangial proliferative glomerulonephritis, leukocytosis, thrombocytosis, anemia and abnormalities of blood chemical parameters have developed in accordance with the elevation of serum IL-6, and 50% of mice have died of renal failure by 18 weeks of age. In contrast, the treatment with MR16-1 prevented all these symptoms and prolonged the lifetime of the majority of the mice. Thus, the constitutive overexpression of IL-6 caused various disorders, and the treatment with anti-IL-6R mAb completely prevented from these symptoms. These results clearly confirm that IL-6 indeed plays an essential role in the pathogenesis of a variety of disorders. Furthermore, anti-IL-6R mAb could provide novel therapy for Castleman's disease and MR16-1 should be a useful tool to estimate therapeutic potential of IL-6 antagonists in a variety of murine models for human disease.