重组人ALR单克隆抗体的制备、鉴定和初步应用

目的 制备抗重组人肝再生增强因子(ALR)的单克隆抗体(McAb)。方法 以基因重组人ALR二聚体(drhALR)为抗原,免疫Balb／c小鼠,制备单克隆抗体。用常规法制备抗ALR单体(mrhALR)多抗,建立夹心ELISA法测定ALR,初步确定ALR在小鼠组织的分布;用荧光免疫方法对胎鼠各组织进行ALR检测。结果 筛选出3株稳定分泌抗drhALR的单抗杂交瘤细胞株,免疫球蛋白亚类分别为IgG1、IgG2a、IgG2b,特异性高,用于rhALR的免疫印迹及ELISA检测,可同时识别rhALR单体(mrhALR)和二聚体(drhALR);夹心ELISA法检测小鼠肝、肾、脾提取液ALR含量与鼠龄无关,各脏器的ALR含量不同,分别为肝0．36μg/g,肾0．11μg/g,脾0．05μg/g;免疫荧光检测胎鼠,各器官有不同程度ALR的存在,无器官特异性。结论 用drhALR为抗原制备的抗rhALR单克隆抗体,能够用于ALR的体内外免疫学检测,为rhALR及生化提取ALR的药理学研究及质量标准奠定了基础。     

Dendritic cells in host response to biologic scaffolds

Tissue regeneration and repair require a highly complex and orchestrated series of events that require inflammation, but can be compromised when inflammation is excessive or becomes chronic. Macrophages are one of the first cells to contact and respond to implanted materials, and mediate the inflammatory response. The series of events following macrophage association with biomaterials has been well-studied. Dendritic cells (DCs) also directly interact with biomaterials, are critical for specific immune responses, and can be activated in response to interactions with biomaterials. Yet, much less is known about the responses by DCs. This review discusses what we know about DC response to biomaterials, the underlying mechanisms involved, and how DCs can be influenced by the macrophage response to biomaterials. Lastly, I will discuss how biomaterials can be manipulated to enhance or suppress DC function to promote a specific desirable immune response – a major goal for implantable biologically active therapeutics.     

Microbial and human heat shock proteins as ‘danger signals’ in sarcoidosis

In the light of the Matzinger’s model of immune response, human heat shock proteins (HSPs) as main ‘danger signals’ (tissue damage-associated molecular patterns-DAMPs) or/and microbial HSPs as pathogen-associated molecular patterns (PAMPs) recognized by pattern recognition receptors (PRR), may induce sarcoid granuloma by both infectious and non-infectious factors in genetically different predisposed host. Regarding infectious causes of sarcoid models, low-virulence strains of, e.g. mycobacteria and propionibacteria recognized through changed PRR and persisting in altered host phagocytes, generate increased release of both human and microbial HSPs with their molecular and functional homology. High chronic spread of human and microbial HSPs altering cytokines, co-stimulatory molecules, and Tregs expression, apoptosis, oxidative stress, induces the autoimmunity, considered in sarcoidosis. Regarding non-infectious causes of sarcoidosis, human HSPs may be released at high levels during chronic low-grade exposure to misfolding amyloid precursor protein in stressed cells, phagocyted metal fumes, pigments with/without aluminum in tattoos, and due to heat shock in firefighters. Therefore, human HSPs as DAMPs and/or microbial HSPs as PAMPs produced as a result of non-infectious and infectious factors may induce different models of sarcoidosis, depending on the genetic background of the host. The number/expression of PRRs/ligands may influence the occurrence of sarcoidosis in particular organs.     

Anxiété : facteur prédictif d’échec du bloc axillaire sous neurostimulation ?

Objective

To evaluate the impact of the anxiety level using Spielberger test on axillary block success.

Study design

Prospective double-blind study.

Patients and methods

An axillary brachial plexus block was performed with a nerve stimulator for all patients undergoing elective or emergency upper limb surgery. Spielberger test result was blinded for both patient and anaesthesiologist performing the block. Time to perform the block (minutes) was measured. Anxiety and pain scores were assessed, using a numeric scale (NS), at different time. Successful block was defined as complete sensory blockade combined with painless during surgical incision. Data were compared using Spearman test and multivariate logistical regression analysis.

Results

Patients (184) were included (elective surgery = 62%; emergency = 38%). Failure rate was 10%. On multivariate logistical regression analysis, time to perform the block and NS anxiety score before starting the block were associated with block failure. Spielberger score correlated with NS anxiety score before puncture (Rho = 0,586, p < 10⁻⁴). Anxiety level was increased in emergency context.

Conclusion

Patient's anxiety level before axillary brachial plexus block is a risk factor of failure, especially in emergency condition. We suggest anesthesiologists to evaluate patient anxiety prior to block performance. A specific anxiolytic treatment may be recommend in some cases.     

大鼠睾丸组织中ALR的研究

目的:研究大鼠睾丸组织中肝再生增强因子(ALR)的表达情况,探讨睾丸ALR与生殖的关系.方法:取幼年、成年、老年大鼠睾丸组织以及部分肝切除(PH)术前、术后12h、24h、48h大鼠肝脏、睾丸组织进行免疫组化,了解大鼠睾丸组织中ALR的表达.结果:睾丸组织中ALR广泛存在于间质细胞、支持细胞和生精细胞,其中尤以精原细胞、精母细胞表达最强.不同年龄段ALR的表达不一样,幼年最强、成年次之、老年最弱.70%PH后肝脏ALR表达增强,24h达峰值,睾丸组织中ALR表达无明显变化.结论:睾丸组织有较强ALR表达,不同脏器的ALR作用具有脏器特异性,ALR与睾丸的发育以及精子生成及成熟密切相关.     

Genetic Recombinant Expression and Characterization of Human Augmenter of Liver Regeneration

Aims To establish a highly effective prokaryotic recombinant expression system for human augmenter of liver regeneration (hALR) and to characterize the recombinant hALR both in vitro and in vivo. Methods ALR cDNA was synthesized and inserted into expression vector pET28a+, the recombinant plasmid was transformed into BL21, and expression of hALR was induced by IPTG. Recombinant hALR (rhALR) was purified by sequential detergent wash, enterokinase (EK) digestion, gel-filtration, and chelating chromatography. The rhALR was identified by SDS-PAGE, immunoblotting, MALDI-TOF-MS, and N-terminal sequencer. Cell proliferative effect of rhALR on human hepatocytes was analyzed by MTT. The protective effect of rhALR on liver function was observed on CCl₄-induced intoxicated mice. Results Recombinant expression plasmid of ALR [pET28(a+)-hALR] was confirmed by restriction enzyme digestion and DNA sequencing. The expressed rhALR constituted 30% of total bacterial protein. Molecular weight was 15,029 for monomer and 30,136 for dimer by mass determination. N-terminal was M-R-T-Q-Q, exactly the same as anticipated for hALR. The purified protein migrating at about 15 KD showed excellent antigenicity in immunoblotting. The rhALR also showed a strong stimulative effect on hepatocyte proliferation. ALT and AST levels, liver histological structure, as well as the survival rate of CCl₄-intoxicated mice were significantly improved when rALR was administrated at 40 μg/kg or 200 μg/kg. Conclusions The rhALR is successfully expressed highly effectively with anticipated MW, N-terminal, and antigenicity. It could play an important role in relieving acute hepatic injury and hepatic failure by promoting hepatic cell proliferation and improving liver function in CCl₄-intoxicated mice. Chun-Fang Gao, Fei Guo Zhou: the authors contributed equally to this work.     

Effect of naked eukaryotic expression plasmid encoding rat augmenter of liver regeneration on acute hepatic injury and hepatic failure in rats

AIM: To study the protective effect of eukaryotic expression plasmid encoding augmenter of liver regeneration (ALR) on acute hepatic injury and hepatic failure in rats. METHODS: The PCR-amplified ALR gene was recombined with pcDNA3 plasmid, and used to treat rats with acute hepatic injury. The rats with acute hepatic injury induced by intraperitoneal injection of 2 mL/kg 50% carbon tetrachloride (CCl4) were randomly divided into saline control group and recombinant pcDNA3-ALR plasmid treatment groups. Recombinant pcDNA3-ALR plasmid DNA (50 or 200 μg/kg) was injected into the rats with acute hepatic injury intraven ously, intraperitoneally, or intravenously and intraperitoneally in combination 4 h after CCl4 administration, respectively. The recombinant plasmid was injected once per 12 h into all treatment groups four times, and the rats were decapitated 12 h after the last injection. Hepatic histopathological alterations were observed after HE staining, the expression of proliferating cell nuclear antigen (PCNA) in liver tissue was detected by immunohistochemical staining, and the level of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) was determined by biochemical method. The recombinant plasmid DNA (200 μg/kg) and saline were intraperitoneally injected into the rats with acute hepatic failure induced by intraperitoneal injection of 4 mL/kg 50% CCl4 after 4 h of CCl4 administration, respectively. Rats living over 96 h were considered as survivals. RESULTS: The sequence of ALR cDNA of recombinant pcDNA3-ALR plasmid was accordant with the reported sequence of rat ALR cDNA. After the rats with acute hepatic injury were treated with recombinant pcDNA3-ALR plasmid, the degree of liver histopathological injury markedly decreased. The pathologic liver tissues, in which hepatic degeneration and necrosis of a small amount of hepatocytes and a large amount of infiltrating inflammatory cells were observed, and they became basically normal in the most effective group after four times of injection of recombinant pcDNA3-ALR plasmid. The indexes of PCNA significantly increased in the recombinant pcDNA3-ALR plasmid treatment groups compared to model group. The level of serum AST and ALT remarkably reduced in recombinant pcDNA3-ALR plasmid treatment groups compared to model group. The results showed that the effect of 200 μg/kg recombinant pcDNA3-ALR plasmid in the rats with acute liver injury was stronger than that of 50 μg/kg pcDNA3-ALR DNA. The effect of intravenous injection of recombinant pcDNA3 ALR plasmid was better. After the rats with acute hepatic failure were treated with recombinant pcDNA3-ALR plasmid, the survival rate (40%) significantly increased in treatment groups compared to control group (15%, P<0.01). CONCLUSION: The ALR gene may play an important role in relieving acute hepatic injury and hepatic failure by promoting hepatic cell proliferation and reducing level of AST and ALT in CCl4-intoxicated rats.