TRAF6调控NLRP3炎症小体信号通路的机制研究

目的:研究肿瘤坏死因子受体相关因子6(TRAF6)对NOD样受体蛋白3(NLRP3)炎症小体信号通路调控作用的机制。方法:利用免疫共沉淀和免疫印迹在HEK-293T细胞中研究TRAF6与NLRP3的相互作用;通过检测乳酸脱氢酶(LDH),在THP-1细胞中研究TRAF6对NLRP3炎症小体信号通路活性的影响。结果:TRAF6通过与NLRP3的相互作用增加NLRP3的稳定性,进而促进NLRP3炎症小体信号通路介导的LDH的释放。结论:TRAF6通过增加NLRP3的稳定性正调控NLRP3炎症小体信号通路。     

炎性小体研究进展

炎性小体是由胞浆内模式识别受体组装的多蛋白复合体,是宿主先天免疫系统的重要组成部分。在细胞应对外界危险信号时,炎性小体能激活半胱天冬酶-1,通过调节白介素-1β和白介素-18等促炎性细胞因子的成熟与释放,参与先天性免疫防御。炎性小体活性异常与人类先天性和获得性炎症性疾病密切相关,炎性小体在免疫应答中具有重要作用。综述了炎性小体的组成、功能、激活方式及其与疾病的相关性。     

汉坦病毒结构蛋白的鉴定及其应用

对差速离心纯化的汉坦病毒R84-1毒株进行了SDS-PAGE和免疫印迹试验。发现有67k和43k两条蛋白区带能与汉坦病毒抗体起反应。经单克隆抗体鉴定,67k多肽可能属病毒囊膜蛋白,43k多肽未定。用免疫印迹法对出血热患者进行检测,初步证明野鼠型感染者具有上述两种蛋白抗原的抗体,大鼠型患者仅具有67k蛋白的抗体,这对出血热患者血清学分型有重要意义。     

蛋白免疫印迹法同时检测大、小分子蛋白的实验条件改进

目的:蛋白免疫印迹法是现代生物医学研究中广泛应用于蛋白定性和半定量分析的实验技术。然而,常规采用传统单一浓度凝胶的蛋白免疫印迹法在应用过程中仍有不足之处,如不能同时检测分子量很大和很小的蛋白,因而有必要探索一种增大凝胶有效分离范围的检测方法。本文提出采用组合凝胶来实现更大范围分子量蛋白的同时检测。方法:比较双浓度的组合凝胶与单一浓度凝胶的分离范围以及分析采用组合凝胶,蛋白免疫印迹法对大、小分子蛋白的检测效果。结果:12%/7.5%组合凝胶和15%/7.5%组合凝胶的分离范围显著大于相应的单一浓度凝胶。通过12%/7.5%组合凝胶,蛋白免疫印迹法同时检测到15-300 k Da范围内的大、小分子蛋白。结论:组合凝胶有助于蛋白免疫印迹法对分子量相差很大的蛋白进行同时检测分析,具有很强的实用性。     

人白细胞介素2受体β(IL-2RB)基因真核表达及与JAK1的相互作用的检测北大核心

[目的]构建含人白介素2受体β(IL-2RB)基因的真核表达质粒p ENTER-IL-2RB-His,并在293T中进行真核表达,并用免疫共沉淀检测JAK1与IL-2RB在细胞内的相互作用。[方法]在Hela细胞中提取人总RNA,通过RT-PCR获得人IL-2RB的基因全长,并将其克隆至真核表达载体p ENTER-His中。经PCR克隆,双酶切、测序鉴定后,将重组质粒p ENTER-IL-2RB-His转染至293T细胞中。免疫印迹法检测不同时间点的IL2RΒ蛋白(24 h、36 h、48 h)在293T细胞中的表达,用免疫共沉淀检测JAK1和IL-2RB蛋白之间的相互作用。[结果]经PCR克隆、双酶切、测序鉴定质粒克隆正确。免疫印迹可见61 k Da的目的蛋白。共同转染JAK1和IL-2RB的质粒,免疫印迹可见分别为133 k Da和61 k Da的目的条带。[结论]成功获得IL-2RB基因全长,成功构建p ENTER-IL-2RB-His真核表达质粒,并在293T细胞中成功表达,随着时间的推移其表达量增高。免疫共沉淀可以检测到JAK1和IL-2RB两者的蛋白相互作用,这为下一步研究JAK1和IL-2RB这一对蛋白的相互作用的作用方式及作用机制奠定了基础。     

川芎嗪对体外培养的胎鼠神经干细胞增殖和分化的影响

目的：探讨川芎嗪（TMP）在体外神经干细胞（NSCs）增殖与分化中的作用。方法：原代提取孕14 d雌性大鼠的胎鼠大脑皮层分离培养,并作免疫荧光染色鉴定,取传代培养第3代的NSCs进行实验。实验分为对照组、β-巯基乙醇阳性对照组、TMP诱导组和TMP+EGTA组（n=4）。采用BrdU法和MTT法观察川芎嗪对NSCs增殖数量的影响,采用蛋白免疫印迹法检测NSCs的分化表达情况。结果：实验成功分离纯化原代NSCs,培养3~5 d可见部分神经球形成,具备典型的NSCs形态并表达NSCs特异抗原巢蛋白;BrdU法和MTT法结果均显示,与对照组和β-巯基乙醇阳性对照组相比,TMP组NSCs增殖数量明显增多（P<0.05）;蛋白免疫印迹结果显示,TMP组和TMP+EGTA组NSCs的神经元分化率明显增高,TMP+EGTA组分化率增高更明显（P<0.05）。结论：TMP能显著增强NSCs的增殖和神经元分化率。减少细胞外Ca²⁺可促进TMP诱导NSCs向神经元分化,Ca²⁺信号在TMP诱导NSCs向神经元分化过程中起重要作用。     

β-淀粉样肽寡聚体的制备及鉴定

[目的]制备β-淀粉样肽(amyloid-βpeptide,Aβ)的可溶性寡聚体。[方法]选取化学合成的Aβ25-35、Aβ1-42及对照肽Aβ35-25,体外37℃孵育7 d制备Aβ寡聚体。采用dot blotting,免疫印迹鉴定Aβ的聚集状态;并通过TUNEL法检测Aβ的神经毒性。[结果]经dot blotting和免疫印迹检测,显示本研究制备获得的Aβ25-35和Aβ1-42均为寡聚体,且Aβ25-35寡聚体的分子量以2 kDa、3 kDa和17 kDa为主,Aβ1-42分子量主要为4kDa和8kDa,而对照肽Aβ35-25并不能形成寡聚体。TUNEL法则显示Aβ25-35寡聚体可以引起SH-SY5Y细胞凋亡。[结论]在体外成功建立制备Aβ寡聚体的方法,且通过形态学实验证实该Aβ有神经毒性,为后续研究阿尔兹海默症的致病机制奠定实验基础。     

炎症小体的研究进展概述CSCD

炎症小体(inflammasome)是一种多蛋白质复合物,它的组装可以导致Caspase-1的激活,并促进炎性细胞因子白介素-1β(interleukin-1β,IL-1β)和白介素-18(interleukin-18,IL-18)的成熟与释放;同时,激活的Caspase-1还可以引发细胞焦亡(pyroptosis)。炎症小体在应答病原体相关分子模式(pathogenassociated molecular pattern,PAMP)或者危险相关分子模式(danger-associated molecular pattern,DAMP)的刺激中扮演着重要角色。炎症小体的异常会导致多种自发炎症及自身免疫性疾病的发生,但是关于其调控活化机制仍有诸多不明,相关研究有待进一步深入。     

NLRP3炎症小体与心脏骤停复苏后心肌缺血再灌注损伤的研究进展

心脏骤停已成为世界范围内的重大公共卫生问题,及时有效的心肺复苏可以挽救生命,改善心脏骤停患者的预后。尽管心肺复苏技术取得了进步,但与心脏骤停相关的死亡率仍然很高。NLRP3炎症小体是细胞内多种蛋白质构成的复合物,在先天免疫中起着重要作用。组织损伤后,NLRP3炎症小体激活产生大量细胞因子如白细胞介素(IL)-1β和IL-18,最终导致炎症性细胞死亡(细胞焦亡)。虽然适度的炎症反应有利于损伤组织愈合,但过度的NLRP3炎症小体激活会产生不利影响。NLRP3炎症小体在大量心血管疾病(cardiovascular diseases, CVDs)中发挥关键作用。心脏骤停和复苏后缺血再灌注损伤(I/R损伤)可以通过各种信号通路激活NLRP3炎症小体。抑制NLRP3炎症小体活性可以改善心脏骤停和复苏后缺血再灌注损伤。该文将讨论NLRP3炎症小体在心脏骤停和复苏过程中对细胞损伤的作用,同时针对抑制NLRP3炎症小体激活,改善心脏骤停和复苏后缺血再灌注损伤的治疗方法进行系统阐述。     

人细小病毒B19基因组编码蛋白真核表达质粒的构建与鉴定

目的:构建人细小病毒B19(B19V)基因组编码蛋白的真核表达质粒,并对其表达进行鉴定,为研究B19V蛋白功能提供支持。方法:设计并合成B19V基因组编码蛋白VP1、VP2、NS1、11k Da、X、7.5k Da和VP1独特区域(VP1u)的PCR扩增引物,以B19V感染性克隆p B19-M20为模板,PCR扩增基因组编码蛋白的核苷酸序列,将其插入真核表达载体pcDNA3.1(+)-HA-Flag,并进行酶切鉴定和核苷酸序列测定;将鉴定正确的重组质粒转染HEK293T细胞,通过免疫印迹分别检测病毒蛋白的表达情况。结果:构建了B19V 7种蛋白的真核表达质粒,免疫印迹鉴定表明均可表达相应的目的蛋白。结论:构建了B19V 7种蛋白的真核表达质粒,为深入研究B19V蛋白功能及其在致病机制中的作用奠定了基础。     

Exosomes with immune modulatory features are present in human breast milk

Breast milk is a complex liquid with immune-competent cells and soluble proteins that provide immunity to the infant and affect the maturation of the infant's immune system. Exosomes are nanovesicles (30-100 nm) with an endosome-derived limiting membrane secreted by a diverse range of cell types. Because exosomes carry immunorelevant structures, they are suggested to participate in directing the immune response. We hypothesized that human breast milk contain exosomes, which may be important for the development of the infant's immune system. We isolated vesicles from the human colostrum and mature breast milk by ultracentrifugations and/or immuno-isolation on paramagnetic beads. We found that the vesicles displayed a typical exosome-like size and morphology as analyzed by electron microscopy. Furthermore, they floated at a density between 1.10 and 1.18 g/ml in a sucrose gradient, corresponding to the known density of exosomes. In addition, MHC classes I and II, CD63, CD81, and CD86 were detected on the vesicles by flow cytometry. Western blot and mass spectrometry further confirmed the presence of several exosome-associated molecules. Functional analysis revealed that the vesicle preparation inhibited anti-CD3-induced IL-2 and IFN-gamma production from allogeneic and autologous PBMC. In addition, an increased number of Foxp3(+)CD4(+)CD25(+) T regulatory cells were observed in PBMC incubated with milk vesicle preparations. We conclude that human breast milk contains exosomes with the capacity to influence immune responses.     

Antigenicity analysis of Vibrio harveyi TS-628 strain

Vibrio harveyi,the major causative agent of vibriosis,affects a diverse range of marine cultured organisms over a wide geographical area.However,reports about screening the effective antigen and research on vaccines of V.harveyi are scarce.Flagellin,lipopolysaccharide (LPS) and outer membrane proteins (OMP) are major immunogenic antigens in many Gram-negative bacteria.In this study,the flagellin,OMP and LPS of the V.harveyi TS-628 strain isolated from infected groupers were extracted and Western blot analysis was used to detect the antigenicity of these extractions.Results of the Western blot assay reveal that there are four positive flagellin bands:35 kDa,38 kDa,43 kDa,and 52 kDa,of which the 43 kDa and 52 kDa bands displayed the strongest positive reaction.There are five positive OMP bands about 35 kDa,38 kDa,43 kDa,47 kDa,and 52 kDa,of which the 43 kDa appeared to have the strongest positive reaction although the other four proteins also displayed strong reactions.However,LPS is Western blot-negative.These results indicate that the 43 kDa and 52 kDa flagellin and OMP of size 43 kDa,52 kDa can be candidates for developing vaccines against V.harveyi.     

The Ether Lipid Precursor Hexadecylglycerol Stimulates the Release and Changes the Composition of Exosomes Derived from PC-3 Cells

Exosomes are vesicles released by cells after fusion of multivesicular bodies with the plasma membrane. In this study, we have investigated whether ether lipids affect the release of exosomes in PC-3 cells. To increase the cellular levels of ether lipids, the ether lipid precursor hexadecylglycerol was added to cells. Lipidomic analysis showed that this compound was in fact able to double the cellular levels of ether lipids in these cells. Furthermore, increased levels of ether lipids were also found in exosomes released by cells containing high levels of these lipids. Interestingly, as measured by nanoparticle tracking analysis, cells containing high levels of ether lipids released more exosomes than control cells, and these exosomes were similar in size to control exosomes. Moreover, silver staining and Western blot analyses showed that the protein composition of exosomes released in the presence of hexadecylglycerol was changed; the levels of some proteins were increased, and the levels of others were reduced. In conclusion, this study clearly shows that an increase in cellular ether lipids is associated with changes in the release and composition of exosomes.     

Induction of exosome release in primary B cells stimulated via CD40 and the IL-4 receptor

Exosomes are lipid-bound nanovesicles formed by inward budding of the endosomal membrane and released following fusion of the endosomal limiting membrane with the plasma membrane. We show here that primary leukocytes do not release exosomes unless subjected to potent activation signals, such as cytokine or mitogen stimulation. In particular, high levels of exosomes were released when murine splenic B cells were stimulated via CD40 and the IL-4 receptor. This property was shared by B cells from different anatomic locations, as newly formed marginal zone and follicular B cells were capable of secreting exosomes upon CD40/IL-4 triggering. B cell exosomes expressed high levels of MHC class I, MHC class II, and CD45RA (B220), as well as components of the BCR complex, namely, surface Ig, CD19, and the tetraspanins CD9 and CD81. Ig on the plasma membrane of primary B cells was targeted to the exosome pathway, demonstrating a link between the BCR and this exocytic pathway. IgD and IgM were the predominant Ig isotypes associated with CD40/IL-4 elicited exosomes, though other isotypes (IgA, IgG1, IgG2a/2b, and IgG3) were also detected. Together, these results suggest that exosome release is not constitutive activity of B cells, but may be induced following cell: cell signaling.     

Proteomic analysis of melanoma-derived exosomes by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry

Exosomes are 40-100 nm vesicles released by numerous cell types and are thought to have a variety of roles depending on their origin. Exosomes derived from antigen presenting cells have been shown to be capable of initiating immune responses in vivo and eradicating established tumours in murine models. Tumour-derived exosomes can be utilised as a source of tumour antigen for cross-priming to T-cells and are thus of interest for use in anti-tumour immunotherapy. Further exploration into the protein composition of exosomes may increase our understanding of their potential roles in vivo and this study has examined the proteome of exosomes purified from cell supernatants of the melanoma cell lines MeWo and SK-MEL-28. The vesicular nature and size (30-100 nm) of the purified exosomes was confirmed by electron microscopy and sucrose density gradient centrifugation. Western blotting demonstrated the absence of calnexin and cytochrome c, verifying the purity of the exosome preparations, as well as enrichment of MHC class I and the tumour-associated antigens Mart-1 and Mel-CAM. The two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) protein profiles of exosomes from the two cell lines were highly comparable and strikingly different from the profiles of the total cell lysates. Mass spectrometric sequencing identified proteins present in 49 protein spots in the exosome lysates. Several of these have been identified previously in exosomes but some are novel, including p120 catenin, radixin, and immunoglobulin superfamily member 8 (PGRL). Proteins present in whole-cell lysates that were significantly reduced or excluded from exosomes were also identified and included several mitochondrial and lysosomal proteins, again confirming the proposed endosomal origin of exosomes. This study presents a starting point for future more in-depth protein studies of tumour-derived exosomes which will aid the understanding of their biogenesis and targeting for use in anti-tumour immunotherapy protocols.     

Antigenicity analysis of Vibrio harveyi TS-628 strain

Vibrio harveyi, the major causative agent of vibriosis, affects a diverse range of marine cultured organisms over a wide geographical area. However, reports about screening the effective antigen and research on vaccines of V. harveyi are scarce. Flagellin, lipopolysaccharide (LPS) and outer membrane proteins (OMP) are major immunogenic antigens in many Gram-negative bacteria. In this study, the flagellin, OMP and LPS of the V. harveyi TS-628 strain isolated from infected groupers were extracted and Western blot analysis was used to detect the antigenicity of these extractions. Results of the Western blot assay reveal that there are four positive flagellin bands: 35 kDa, 38 kDa, 43 kDa, and 52 kDa, of which the 43 kDa and 52 kDa bands displayed the strongest positive reaction. There are five positive OMP bands about 35 kDa, 38 kDa, 43 kDa, 47 kDa, and 52 kDa, of which the 43 kDa appeared to have the strongest positive reaction although the other four proteins also displayed strong reactions. However, LPS is Western blot-negative. These results indicate that the 43 kDa and 52 kDa flagellin and OMP of size 43 kDa, 52 kDa can be candidates for developing vaccines against V. harveyi. Translated from Journal of Xiamen University (Natural Science), 2006, 45(3): 393–396 [译自: 厦门大学学报 (自然科学版)]     

Exosomes as potent cell-free peptide-based vaccine. I. Dendritic cell-derived exosomes transfer functional MHC class I/peptide complexes to dendritic cells

Current immunization protocols in cancer patients involve CTL-defined tumor peptides. Mature dendritic cells (DC) are the most potent APCs for the priming of naive CD8(+) T cells, eventually leading to tumor eradication. Because DC can secrete MHC class I-bearing exosomes, we addressed whether exosomes pulsed with synthetic peptides could subserve the DC function consisting in MHC class I-restricted, peptide-specific CTL priming in vitro and in vivo. The priming of CTL restricted by HLA-A2 molecules and specific for melanoma peptides was performed: 1) using in vitro stimulations of total blood lymphocytes with autologous DC pulsed with GMP-manufactured autologous exosomes in a series of normal volunteers; 2) in HLA-A2 transgenic mice (HHD2) using exosomes harboring functional HLA-A2/Mart1 peptide complexes. In this study, we show that: 1). DC release abundant MHC class I/peptide complexes transferred within exosomes to other naive DC for efficient CD8(+) T cell priming in vitro; 2). exosomes require nature's adjuvants (mature DC) to efficiently promote the differentiation of melanoma-specific effector T lymphocytes producing IFN-gamma (Tc1) effector lymphocytes in HLA-A2 transgenic mice (HHD2). These data imply that exosomes might be a transfer mechanism of functional MHC class I/peptide complexes to DC for efficient CTL activation in vivo.     

A new method to precipitate myosin V from rat brain soluble fraction

Myosin can be precipitated from soluble fraction under different assay conditions. This paper describes a new method for precipitating myosin V from rat brain soluble fraction. Brains were homogenized in 50 mM imidazole/HCl buffer, pH 8.0, containing 10 mM EDTA/EGTA, 250 mM sucrose, 1 mM DTT and 1 mM benzamidine, centrifuged at 45000 x g for 40 min and the supernatant was frozen at -20 degrees C. Forty-eight hours later, the supernatant was thawed, centrifuged at 45000 x g for 40 min and the precipitate was washed in 20 mM imidazole buffer pH 8.0. SDS/PAGE analysis showed four polypeptides in the precipitate: 205, 150, 57 and 43 kDa. The precipitate presented high Mg(2+)-ATPase activity, which co-purifies with p205. This polypeptide was recognized by a specific myosin V antibody and was proteolised by calpain, generating two stable polypeptides: p130 and p90. The Mg(2+)-ATPase activity was not stimulated by calcium in both the absence and presence of exogenous calmodulin and the K+/EDTA-ATPase activity represented 25% of the Mg(2+)-ATPase activity. In this work, myosin V from rat brain was precipitated by freezing the soluble fraction and was co-purificated with a 45 kDa polypeptide.     

Trafficking and localization studies of recombinant alpha1, 3- fucosyltransferase VI stably expressed in CHO cells

Peripheral alpha1,3-fucosylation of glycans occurs by the action of either one of five different alpha1,3-fucosyltransferases (Fuc-Ts) cloned to date. Fuc-TVI is one of the alpha1,3-fucosyltransferases which is capable to synthesize selectin ligands. The major alpha1, 3- fucosyltransferase activity in human plasma is encoded by the gene for fucosyltransferase VI, which presumably originates from liver cells. While the sequence, chromosomal localization, and kinetic properties of Fuc-TVI are known, immunocytochemical localization and trafficking studies have been impossible because of the lack of specific antibodies. Here we report on the development and characterization of a peptide-specific polyclonal antiserum monospecific to Fuc-TVI and an antiserum to purified soluble recombinant Fuc-TVI crossreactive with Fuc-TIII and Fuc-TV. Both antisera were applied for immunodetection in stably transfected CHO cells expressing the full-length form of this enzyme (CHO clone 61/11). Fuc-TVI was found to be a resident protein of the Golgi apparatus. In addition, more than 30% of cell-associated and released enzyme activity was found in the medium. Maturation and release of Fuc-TVI was analyzed in metabolically labeled CHO 61/11 cells followed by immunoprecipitation. Fuc-TVI occurred in two forms of 47 kDa and 43 kDa bands, while the secreted form was detected as a 43 kDa. These two different intracellular forms arose by posttranslational modification, as shown by pulse-chase experiments. Fuc-TVI was released to the supernatant by proteolytic cleavage as a partially endo-H resistant glycoform.      

Developmental regulation of glucosidase I, an enzyme involved in the processing of asparagine-linked glycoproteins in rat mammary gland

Glucosidase I involved in the processing of N-linked glycoproteins was purified to homogeneity from the lactating rat mammary gland. The purified enzyme exhibited a single band at 85 kDa on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Polyclonal antibodies raised against the enzyme recognized a similar band on Western blots and also inhibited the enzyme activity. The enzyme levels gradually increased until the midlactation stage and thereafter declined sharply during the period of postlactation. A similar profile of the levels of immunoreactive glucosidase I was observed. These findings suggest that the accumulation of glucosidase I is modulated as a function of gland ontogeny. The results on hormonal regulation of glucosidase I indicate that the synthesis of the enzyme is stimulated by a combination of insulin, hydrocortisone, and prolactin; additionally, epidermal growth factor may play a role in this regulation. The above observation was substantiated by immunoprecipitation of [35S]methionine-labeled microsomal extracts with anti-glucosidase I antibodies. The immunoprecipitation of soluble extracts from [35S]methionine-labeled tissue with anti-rat alpha-lactalbumin antibodies indicates that these hormones not only stimulate the synthesis of alpha-lactalbumin but also play an important role in its glycosylation.