蛋白质分离纯化方法的研究进展

蛋白质作为生命物质基础之一,存在于自然界中复杂的混合体系中。蛋白质在细胞中的含量极低,将其从复杂体系中分离出来并保持其活性,难度较大,因此分离纯化蛋白质的方法受到生命科学领域广泛关注。本文首先阐述了蛋白质的预处理,其次阐述了蛋白质分离纯化的各种方法如沉淀、层析、离心等,重点阐述了蛋白质新纯化的各种原理,以及每种方法的适用范围。有助大家更全面、更彻底的了解蛋白纯化技术的发展,以期为今后开展蛋白质的制备及应用提供一定理论依据和实验指导。     

人类白细胞抗原半相合供者外周血细胞因子诱导的杀伤细胞治疗晚期原发性肝癌二例

目的 观察半相合供者细胞因子诱导的杀伤细胞(CIK)治疗晚期肝癌的临床疗效.方法 2 例无法手术且无法耐受化疗的晚期肝癌患者分别选取人类白细胞抗原(HLA)半相合健康供者体外培养CIK,14 d 后分次回输给相应患者,观察患者治疗前后瘤体大小、肿瘤标志物、免疫指标、临床症状、生活质量和卡氏评分等的变化.结果 2 例患者...     

Temporal and spatial variability of chlorophyll <Emphasis Type="Italic">a</Emphasis> concentration in Lake Taihu using MODIS time-series data

In order to predict the distribution of chlorophyll a synoptically in Lake Taihu from 2006 to 2008, a common empirical algorithm was developed to relate time series chlorophyll a concentrations in the lake to reflectance derived as a function of band 2 MODIS data (r ² = 0.907, n = 145) using time series from 2005. The empirical model was further validated with chlorophyll a data from a 2008 to 2009 dataset, with RMSE < 7.48 μg l⁻¹ and r ² = 0.978. The seasonal and inter-annual variability of the surface chlorophyll a concentration from 2006 to 2008 was then examined using Empirical Orthogonal Function (EOF) analysis. The results revealed that the first four modes were significant, explaining 54.0% of the total chlorophyll a variance, and indicated that during the summer, algal blooms always occur in the northern bays, Meiliang Bay and Gonghu Bay, while they occur along the southwestern lakeshore during early summer, fall, and even early winter. The inter-annual variance analysis showed that the duration of algal blooms was from April to December of 2007, which was different from the bloom periods in 2006 and 2008. The results of EOF analysis show its potential for long-term integrated lake monitoring, not only in Lake Taihu but also in other large lakes threatened by accelerating eutrophication.     

基因谱系示踪技术揭示小鼠Tbx18+心脏祖细胞向心系细胞分化的潜能

心脏祖细胞(cardiac progenitor cells,CPCs)的研究对阐明先天性心脏病的机制及治疗心血管疾病具有重要意义.哺乳动物的心脏组织由多种不同CPCs分化形成.转录因子Tbx18在发育中的心外膜中表达,对心脏的发育形成起重要的调节作用.为了在组织及活体细胞水平检测和阐明Tbx18+CPC的分化潜能,应用Cre-LoxP系统建立Tbx18+CPCs基因命运谱系示踪模型:Tbx18-Cre/Rosa26R-EYFP和Tbx18-Cre/Rosa26R-LacZ双杂合基因敲入小鼠.该双杂合基因敲入小鼠通过Cre的表达能有效地示踪Tbx18+细胞在胚胎和成年小鼠中的分化命运.Tbx18-Cre/Rosa26R-EYFP双杂合小鼠心脏能非常容易地利用流式细胞分选系统(FACS)分离出YFP+细胞,也可在倒置共聚焦显微镜下观察.应用X-gal染色分析其表达模式,揭示Tbx18命运谱系参与心房肌、室间隔、心室肌、冠状动脉、瓣膜等的形成.应用免疫荧光技术初步揭示Tbx18+CPCs向心脏肌钙蛋白T(cTNT)阳性心肌细胞和平滑肌肌球蛋白重链11(MYH11)阳性血管平滑肌细胞分化的潜能.心脏是一个由多种肌肉和非肌肉组织细胞构成的复杂器官.推测Tbx18可能在心脏祖细胞向肌源性细胞分化的信号通路中起重要调节作用.在上述研究中应用基因谱系示踪技术,验证Tbx18可作为一类CPCs的标志,为更深入揭示心脏祖细胞向心系细胞的分化潜能打下基础.     

Production of antioxidant and antitumor metabolites by submerged cultures of <Emphasis Type="Italic">Inonotus obliquus</Emphasis> cocultured with <Emphasis Type="Italic">Phellinus punctatus</Emphasis>

While Inonotus obliquus produces a diverse range of bioactive metabolites in its natural habitats, it accumulates less in its submerged cultures. We show here that coculture of I. obliquus with Phellinus punctatus resulted in less production of mycelial biomass but an increased accumulation of phenolic compounds, melanins, and lanostane-type triterpenoids. Metabolites increased in production by coculture include phelligridin C, phelligridin H, methyl inoscavin A, inoscavin C, inoscavin B, davallialactone, methyl davallialactone, foscoparianol D, 21,24-cyclopentalanosta-3β,21,25-triol-8-en, lanosta-7,9(11),23-triene-3β,22,25-triol, and inotodisaccharide and melanins. Metabolites from coculture also showed an increased potential for scavenging free radicals and inhibiting the proliferation of HeLa 229 cells. Davallialactone, methyl davallialactone, and minor phenolic components are the major contributors for scavenging DPPH and hydroxyl radical in monoculture, and phelligridin C, phelligridin H, methyl inoscavin A, inoscavin C, methyl davallialactone, foscoparianol D, and inotodisaccharide are those for scavenging the tested radicals in coculture. Lanostane-type triterpenoids indicated limited roles in scavenging free radicals. Nearly all the detected metabolites correlate positively with inhibiting proliferation of HeLa 229 cells. Thus, coculture of I. obliquus with other fungi seems to be a cost-effective strategy for upregulating biosynthesis of bioactive metabolites.     

Pretreatment with H_2O_2 Alleviates Aluminum-induced Oxidative Stress in Wheat Seedlings

Hydrogen peroxide(H2O2) is a key reactive oxygen species(ROS) in signal transduction pathways leading to activation of plant defenses against biotic and abiotic stresses.In this study,we investigated the effects of H2O2 pretreatment on aluminum(Al) induced antioxidant responses in root tips of two wheat(Triticum aestivum L.) genotypes,Yangmai-5(Al-sensitive) and Jian-864(Al-tolerant).Al increased accumulation of H2O2 and O2r· leading to more predominant lipid peroxidation,programmed cell death and root elon...     

The Vacuolar Na+/H+ Antiporter Gene SsNHX1 from the Halophyte Salsola soda Confers Salt Tolerance in Transgenic Alfalfa (Medicago sativa L.)

A putative vacuolar Na⁺/H⁺ antiporter gene (SsNHX1) was isolated from the halophyte Salsola soda using the rapid amplification of cDNA ends method. Highly conserved regions of plant vacuolar Na⁺/H⁺ antiporter, including amiloride-binding domain, NHE (Na⁺/H⁺ exchange) domain, and 12 transmembrane segments, were found in the deduced amino acid sequence of SsNHX1. Multiple alignments of vacuolar Na⁺/H⁺ antiporters showed that SsNHX1 shared high identity with other plant vacuolar Na⁺/H⁺ antiporters. Phylogenetic relationship analysis indicated that SsNHX1 was clustered into the vacuolar Na⁺/H⁺ antiporter group. Taken together, these results suggest that SsNHX1 is a new member of the vacuolar Na⁺/H⁺ antiporter family. The effective expression of SsNHX1 in alfalfa (Medicago sativa L.) enhanced the salt tolerance of transgenic alfalfa which could grow in high concentrations of NaCl (up to 400 mM) over 50 days. This was the highest level of salt tolerance reported in transgenic plants. A further analysis of the physiological characteristics of transgenic and wild-type plants, including the Na⁺ and K⁺ contents, superoxide dismutase activity, the rate of electrolyte leakage, and the proline content, showed that large amounts of Na⁺ in the cytoplasm of leaves were transported into vacuoles by the exogenous Na⁺/H⁺ antiporter, which averted the toxic effects of Na⁺ to the cell of transgenic alfalfa.     

In vitro screening system for hepatotoxicity: Comparison of bone‐marrow‐derived mesenchymal stem cells and Placenta‐derived stem cells

Stem cells have unique properties such as self‐renewal, plasticity to generate various cell types, and availability of cells of human origin. The characteristics are attentive in the toxicity screening against chemical toxicants. Placenta‐derived stem cells (PDSCs) have been spotlighted as a new cell source in stem cell research recently because they are characterized by their capacity to differentiate into multilineages. However, the use of PDSCs as an in vitro screening model for potential drug candidates has not yet been studied. Here, we analyzed the potentials for bone‐marrow‐derived mesenchymal stem (BM‐MSCs), which is a representative adult stem cells and PDSCs as an in vitro hepatotoxicity screening system, using well‐known hepatotoxicants. BM‐MSCs and PDSCs were analyzed to the potential for hepatogenic differentiation and were cultured with different concentrations of hepatotoxicants for time courses. The viability and ATP‐binding cassette (ABC) transporters were measured by the MTT assay and RT‐PCR, respectively. The sensitivities of PDSCs to hepatotoxicants are more sensitive than those of BM‐MSCs. The viability (IC₅₀) to in PDSCs was less than that of BM‐MSCs after 48 and 72 h (P < 0.05) of CCl₄ exposure. The toxicities of CCl₄ were decreased by fourfold in hepatogenic differentiation inducing PDSCs compared to the undifferentiated cells. The alteration of ABCGs was observed in PDSCs during differentiation. These findings suggest that the naïve PDSCs expressing ABCGs can be used as a source for in vitro screening system as well as the expression patterns of ABCG1 and ABCG2 might be involved in the sensitivity of PDSCs to hepatotoxicants. J. Cell. Biochem. 112: 49–58, 2011. © 2010 Wiley‐Liss, Inc.     

The specific binding of peptide ligands to cardiomyocytes derived from mouse embryonic stem cells

Purification of pluripotent stem cell (PSC)‐derived cardiomyocytes is critical for the application of cardiomyocytes both in clinical and basic research. Finding a specific cell marker is a promising method for purifying induced cells. The present study employed phage display technology to search for particular cell markers that could bind specifically to PSC‐derived cardiomyocytes. After three rounds of biopanning, several peptides were obtained. The ELISA results show the no. 3 sequence peptide (QPFTTSLTPPAR), and other four sequences having a consensus motif [SS(Q)PPQ(S)], no. 9, 11, 14, and 10, have relatively high affinity and specificity to cardiomyocytes. Immunofluorescence confirmed that the selected peptides could bind specifically to the PSC‐derived cardiomyocytes. Competition tests with chemically synthesized peptides revealed the binding ability was caused by the peptide itself. Western blot analysis proved the phages were both bound to two 17 kDa cardiomyocyte membrane proteins and the no. 9 sequence showed a 55 kDa protein that was not observed in the no. 3 sequence. These results suggest that the selected peptides specifically target receptors on PSC‐derived cardiomyocyte membranes. The results will pave the way for further studies of cell surface markers and their applications, such as labeling, purification, and as vehicles for drug delivery. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.