血红蛋白氧载体的研究目标及新进展

文题释义：血红蛋白氧载体：以血红蛋白为原料,通过交联、聚合、偶联等化学修饰或者将其封装在人工合成的膜中,得到一种具有运载氧气能力的红细胞替代物。 红细胞替代物：通过模拟天然红细胞生物学功能的氧载体,主要包括基于全氟碳的乳剂、含铁(Fe²⁺)卟啉和基于血红蛋白的氧载体。与红细胞相比,红细胞替代物具有降低感染风险、延长储存时间、在使用时无需配型等优点。 背景：血红蛋白氧载体作为红细胞替代物之一,已研究40余年,至今共有3代不同的血红蛋白氧载体。第1代和第2代主要是对血红蛋白进行化学修饰,而第3代主要是将血红蛋白封装在合成膜中。 目的：综述国内外血红蛋白氧载体研究的新进展。 方法：以“hemoglobin-based oxygen carriers,red blood cell substitutes,artificial oxygen carriers ,artificial blood”“血红蛋白氧载体,红细胞替代物,人工氧载体,人工血液”为检索词,检索2000至2019年PubMed、CNKI中国期刊全文数据库、万方数据库中收录的血红蛋白氧载体制备方法的相关文献,进行总结分析。 结果与结论：①胎儿、无脊椎动物、爬行动物的血红蛋白以及β-亚基突变的血红蛋白在制备血红蛋白氧载体上有一定优点;②关于血红蛋白的修饰和封装策略有了新的进展;③联合使用其他药物能够降低血红蛋白氧载体的血管毒性;④血红蛋白氧载体在应用上有了新方向,但是新型血红蛋白氧载体作为红细胞替代物领域的研究,在未来还需要更多的动物实验和临床试验。 ORCID: 0000-0003-4004-115X(杨康) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

黄芩苷调控自噬保护肠黏膜屏障干预肠道急性移植物抗宿主病

文题释义：移植物抗宿主病：是由于移植后异体供者移植物中的T淋巴细胞,经受者发动的一系列“细胞因子风暴”刺激,显著增强了其对受者抗原的免疫反应,以受者靶细胞为目标发动细胞毒攻击,其中皮肤、肝及肠道是主要的靶目标。根据移植物抗宿主病在移植后发生的时间,如在100 d内发生者称为急性移植物抗宿主病,在100 d后发生者称为慢性移植物抗宿主病。黄芩苷：黄芩汤的君药黄芩,《神农本草经》谓可治“肠辟泄利”,性苦寒,可清肺胃胆及大肠经之湿热,黄芩苷是黄芩中提取的一种黄酮类化合物,具有抑菌、清除氧自由基等作用,减轻蛋白酶与炎性因子对组织的损害。 摘要背景：急性移植物抗宿主病是造血干细胞移植后早期最容易致死的并发症之一,如何干预急性移植物抗宿主病病程是个热点问题。目的：探讨黄芩苷调控自噬干预肠道急性移植物抗宿主病的机制。方法：⁶⁰Co照射CB6F1小鼠4 h,立即尾静脉输注Balb/c小鼠单个核细胞悬液(骨髓+脾),建立单倍体造血干细胞移植模型。造模后当天开始模型组灌胃生理盐水,治疗组灌胃黄芩苷,剂量为30 mg/(kg·d),共14 d。治疗后进行小鼠急性移植物抗宿主病临床评分、小肠黏膜急性移植物抗宿主病病理评级,透射电镜观察小肠黏膜自噬泡结构,流式细胞仪检测小肠黏膜上皮细胞活性氧水平,Western blot检测自噬相关蛋白LC3Ⅱ、LC3-Ⅰ、Beclin1的表达。结果与结论：①治疗组临床急性移植物抗宿主病评分、小肠黏膜病理评级明显优于模型组;②透射电镜下模型组存在自噬泡但线粒体结构毁坏严重,治疗组自噬泡增多且线粒体结构相对完整;③治疗组小鼠小肠黏膜上皮细胞活性氧水平低于模型组(P < 0.01),治疗组LC3Ⅱ/Ⅰ、Beclin1的表达水平明显高于模型组(P < 0.01);④结果表明,黄芩苷可以通过降低活性氧的产生,提高小肠黏膜细胞的自噬水平,保护肠黏膜屏障,进而降低移植后肠道急性移植物抗宿主病的发病率。ORCID: 0000-0002-8771-5898(崔兴) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

不同高压氧吸氧时间对脑出血大鼠血肿周围B淋巴细胞瘤-2基因和BCL2-Associated X蛋白表达的影响

目的 观察不同高压氧（HBO）吸氧时间对脑出血（ICH）大鼠血肿周围B淋巴细胞瘤-2基因(Bcl-2)和BCL2-Associated X蛋白(Bax)表达的影响,以期探索HBO 治疗脑出血的治疗机制和最佳吸氧时间。 方法 选取Wistar大鼠130只,其中正常大鼠5只（正常组）,造模大鼠125只（胶原酶诱导法建立ICH模型）,按随机数字表法将造模大鼠随机分为对照组（无高压氧干预）25只和实验组（高压氧干预）100只,实验组根据吸氧时间的不同再按随机数字表法分为HBO 40 min组,HBO 60 min组,HBO 80 min组,HBO 100 min组,共4个亚组,每组25只大鼠。4个实验组亚组大鼠分别于ICH后6 h后行对应的HBO治疗,每日治疗1次,于治疗第1、3、5、7、14天后每个时间点各处死组内5只大鼠（对照组于相同时间点各处死组内5只大鼠,正常组于入组当天处死并取材）。采用免疫组化法检测各组大鼠各取材时间点血肿周边脑组织中Bax和Bcl-2蛋白的表达情况。 结果 HBO 40 min组、HBO 60 min组、HBO 80 min组、HBO 100 min组各时间点的Bax表达较对照组相同时间点均显著减少,差异均有统计学意义（P<0.05）。其中HBO 60 min组和HBO 80 min组的Bax表达分别与HBO40 min组和HBO 100 min组相同时间点比较,差异均有统计学意义（P<0.05）。造模成功3 d、5 d、7 d、14 d后,HBO 40 min组和HBO 60 min组的Bcl-2表达分别与HBO 80 min组和HBO 100 min组同时间点比较,差异均有统计学意义（P<0.05）。HBO 80 min组在造模成功7 d和14 d后的Bcl-2/Bax比值分别为1.5792和1.5733,与HBO 40 min组、HBO 60 min组以及HBO 100 min组比较,差异均有统计学意义（P<0.05）。 结论 HBO治疗ICH的机制可能与其可以下调出血灶周围Bax的表达水平以及提高Bcl-2的表达水平有关,而HBO治疗ICH的最佳吸氧时间是80 min。     

A genetically-encoded KillerRed protein as an intrinsically generated photosensitizer for photodynamic therapy

Photodynamic therapy (PDT) has received considerable attention as a therapeutic treatment for cancer and other diseases; however, it is frequently accompanied by prolonged phototoxic reaction of the skin due to slow clearance of synthetic photosensitizers (PSs) administered externally. This study was designed to investigate the genetic use of pKillerRed-mem, delivered using complexes of chitosan (CS) and poly(γ-glutamic acid) (γPGA), to intracellularly express a membrane-targeted KillerRed protein that can be used as a potential PS for PDT. Following transfection with CS/pKillerRed/γPGA complexes, a red fluorescence protein of KillerRed was clearly seen at the cellular membranes. When exposed to green-light irradiation, the KillerRed-positive cells produced an excessive amount of reactive oxygen species (ROS) in a time-dependent manner. Data from viability assays indicate that ROS have an important role in mediating KillerRed-induced cytotoxicity, apoptosis, and anti-proliferation, suggesting that KillerRed can be used as an intrinsically generated PS for PDT treatments. Notably, the phototoxic reaction of KillerRed toward cells gradually became negligible over time, presumably because of its intracellular degradability. These experimental results demonstrate that this genetically encoded KillerRed is biodegradable and has potential for PDT-induced destruction of diseased cells.     

Modulation of monocytic leukemia cell function and survival by high gradient magnetic fields and mathematical modeling studies

The influence of spatially modulated high gradient magnetic fields on cellular functions of human THP-1 leukemia cells is studied. We demonstrate that arrays of high-gradient micrometer-sized magnets induce i) cell swelling, ii) prolonged increased ROS production, and iii) inhibit cell proliferation, and iv) elicit apoptosis of THP-1 monocytic leukemia cells in the absence of chemical or biological agents. Mathematical modeling indicates that mechanical stress exerted on the cells by high magnetic gradient forces is responsible for triggering cell swelling and formation of reactive oxygen species followed by apoptosis. We discuss physical aspects of controlling cell functions by focused magnetic gradient forces, i.e. by a noninvasive and nondestructive physical approach.     

The influence of lysosomal stability of silver nanomaterials on their toxicity to human cells

How silver nanomaterials (Ag NMs) could induce toxicity has been debated heatedly by many researchers. We utilized Ag nanoclusters (Ag NCs) with the same size and ligand protection but different core surface speciation. Ag⁺-rich NCs (Ag⁺-R NCs) and their counterpart, the reduced Ag⁰-rich NCs (Ag⁰-R NCs) are synthesized to represent possible dichotomous stages in silver nanomaterial degradation process. Here we show Ag⁰-R NCs induce higher cellular toxicity when compared to Ag⁺-R NCs. This cellular toxicity is brought about via the modulation of reactive oxygen species (ROS) in cells as a result of the more rapid release of Ag species from Ag⁰-R NCs and subsequent oxidation into Ag⁺ in the lysosomal compartment. The weaker Ag⁰-R bond greatly potentiated the release of Ag species in the acidic and enzymatic processes within the lysosomes. Since lysosomes are absent in bacteria, increasing silver nanomaterials stability may lower toxicity in mammalian cells whilst not reducing their efficacy to fight bacteria; this redesign can result in a safer silver nanomaterial.     

Augmented cellular trafficking and endosomal escape of porous silicon nanoparticles via zwitterionic bilayer polymer surface engineering

The development of a stable vehicle with low toxicity, high cellular internalization, efficient endosomal escape, and optimal drug release profile is a key bottleneck in nanomedicine. To overcome all these problems, we have developed a successful layer-by-layer method to covalently conjugate polyethyleneimine (PEI) and poly(methyl vinyl ether-co-maleic acid) (PMVE-MA) copolymer on the surface of undecylenic acid functionalized thermally hydrocarbonized porous silicon nanoparticles (UnTHCPSi NPs), forming a bilayer zwitterionic nanocomposite containing free positive charge groups of hyper-branched PEI disguised by the PMVE-MA polymer. The surface smoothness, charge and hydrophilicity of the developed NPs considerably improved the colloidal and plasma stabilities via enhanced suspensibility and charge repulsion. Furthermore, despite the surface negative charge of the bilayer polymer-conjugated NPs, the cellular trafficking and endosomal escape were significantly increased in both MDA-MB-231 and MCF-7 breast cancer cells. Remarkably, we also showed that the conjugation of surface free amine groups of the highly toxic UnTHCPSi-PEI (Un-P) NPs to the carboxylic groups of PMVE-MA renders acceptable safety features to the system and preserves the endosomal escape properties via proton sponge mechanism of the free available amine groups located inside the hyper-branched PEI layer. Moreover, the double layer protection not only controlled the aggregation of the NPs and reduced the toxicity, but also sustained the drug release of an anticancer drug, methotrexate, with further improved cytotoxicity profile of the drug-loaded particles. These results provide a proof-of-concept evidence that such zwitterionic polymer-based PSi nanocomposites can be extensively used as a promising candidate for cytosolic drug delivery.     

Cellular internalization of doxorubicin loaded star-shaped micelles with hydrophilic zwitterionic sulfobetaine segments

Four arm star-shaped poly(ε-caprolactone)-b-poly((N,N-diethylaminoethyl methacrylate)-r-(N-(3-sulfopropyl)-N-methacryloxyethy-N,N-diethylammoniumbetaine)) (4sPCLDEAS) micelles were loaded with anticancer drug doxorubicin to track their endocytosis in Hela cancer cell line. The effects of mean diameters and surface charges of the drug loaded micelles on the cellular uptake were studied in details. The results demonstrated that the internalization of micelles was both time and energy dependent process. Endocytic pathways including clathrin-mediated endocytosis, caveolae-mediated endocytosis and macropinocytosis were all involved in the internalization; caveolae-mediated endocytosis was the main pathway for the internalization of 4sPCLDEAS micelles. The assays for cell apoptosis and growth inhibition of tumor spheroids identified that these doxorubicin loaded micelles could induce cell apoptosis and inhibit tumor spheroids growth efficiently, which was even equal to free DOX·HCl. This study provided a rational design strategy for fabricating diverse micellar drug delivery systems with high anticancer efficiency.     

Identification and analysis of an intracellular Cu/Zn superoxide dismutase from Sepiella maindroni under stress of Vibrio harveyi and Cd

Superoxide dismutases (SODs) are ubiquitous family of metalloenzymes involved in protecting organisms from excess reactive oxygen species damage. In this paper, a novel intracellular Cu/ZnSOD from Sepiella maindroni (designated as SmSOD) was identified and characterized. The full-length cDNA sequence of SmSOD (GenBank accession No. KF908850) was 709 bp containing an open reading frame (ORF) of 459 bp, encoding 153 amino acid residues peptide with predicted pI/MW (6.02/15.75 kDa), a 131 bp-5′- and 116 bp-3′- untranslated region (UTR). BLASTn analysis and phylogenetic relationship strongly suggested that the sequence shared high similarity with known Cu/Zn SODs. Several highly conserved motifs, including two typical Cu/Zn SOD family domains, two conserved Cu-/Zn-binding sites (H-47, H-49, H-64, H-120 for Cu binding, and H-64, H-72, H-81, D-84 for Zn binding) and intracellular disulfide bond (C-58 and C-146), were also identified in SmSOD. Time-dependent mRNA expression of SmSOD in hepatopancreas was recorded by quantitative real-time RT-PCR after Vibrio harveyi injection and Cd²⁺ exposure. The results indicated that SmSOD was an acute-phase protein involved in the immune responses against pathogens and biological indicator for metal contaminants in aquatic environment.