MTT法检测大鼠外周血淋巴细胞增殖反应探讨与应用

为了更精准简捷地评价机体的细胞免疫状态,采用MTT比色分析法检测大鼠外周血淋巴细胞的增殖能力,并对试验的最适条件进行了探讨,然后将该法应用到Transwell共培养体系中以评价H9N2 AIV感染内皮细胞后对跨内皮淋巴细胞增殖能力的影响。结果表明：在96孔细胞培养板中接种密度为1.25×10⁶个/mL的淋巴细胞于含10% FBS的RPMI 1640培养基中培养的最适时间为48 h。感染病毒后,跨内皮淋巴细胞的增殖明显受抑制;内皮完整状态下,淋巴细胞和病毒共孵育后,活病毒的量降低,而当内皮不存在或被病毒感染后,这一现象消失。结果证明作为机体细胞免疫功能评价指标之一的淋巴细胞增殖能力可采用改良后的MTT法检测。     

钻石海棠茎段的组织培养技术研究

为了建立钻石海棠的组织培养快繁体系,以钻石海棠单芽茎段为外植体,以MS为基础培养基进行组织培养研究,结果表明:5月份为单芽茎段外植体获取的最佳时间,此时污染率和死亡率都低,分别为15.3%和9.4%;最佳丛生芽诱导培养基为MS+6-BA 1.0mg/L+NAA 0.1mg/L,诱导率为60.7%;最佳增殖培养基为MS+6-BA 2.0mg/L+NAA 0.2mg/L,暗培养2周,增殖系数为4.65;生根培养基以1/2MS+IBA0.2mg/L+NAA 0.2mg/L较好,平均根长为6.3cm,生根率为97.2%。     

Effects of nanosilicon dioxide application on in vitro proliferation of apple rootstock

Although silicon (Si) is the second most abundant element of the earth's crust and in soils, it is not listed among the essential elements for plants. However, the beneficial role of Si in stimulating the growth and development of many plant species has been recognized. This study investigated the effects of in vitro application of nanosilicon oxide on growth and proliferation of apple rootstock MM₁₀₆ in tissue culture. The experiment included five levels nanosilicon oxide (0, 25, 50, 100, and 200 mg/L) added to Murashige and Skoog medium. The results showed that using nanosilicon increased in fresh and dry weights, length and number of branches, and chlorophyll in explants with the highest increase being at 100 mg/L. Growth suppression occurred at 200 mg/L. This investigation showed that 100 mg/L silicon oxide can be added to Murashige and Skoog medium for fast growth and proliferation of MM₁₀₆ apple rootstock explants.     

Proteins and Carbohydrates from Red Seaweeds: Evidence for Beneficial Effects on Gut Function and Microbiota

Based on their composition, marine algae, and namely red seaweeds, are good potential functional foods. Intestinal mucosal barrier function refers to the capacity of the intestine to provide adequate containment of luminal microorganisms and molecules. Here, we will first outline the component of seaweeds and will summarize the effects of these on the regulation of mucosal barrier function. Special attention will be paid to unique components of red seaweeds: proteins and derived peptides (e.g., phycobiliproteins, glycoproteins that contain “cellulose binding domains”, phycolectins and the related mycosporine-like amino acids) together with polysaccharides (e.g., floridean starch and sulfated galactans, such as carrageenans, agarans and “dl-hybrid”) and minerals. These compounds have been shown to exert prebiotic effects, to regulate intestinal epithelial cell, macrophage and lymphocyte proliferation and differentiation and to modulate the immune response. Molecular mechanisms of action of peptides and polysaccharides are starting to be elucidated, and evidence indicating the involvement of epidermal growth factor receptor (EGFR), insulin-like growth factor receptor (IGFR), Toll-like receptors (TLR) and signal transduction pathways mediated by protein kinase B (PKB or AKT), nuclear factor-κB (NF-κB) and mitogen activated protein kinases (MAPK) will also be summarized. The need for further research is clear, but in vivo experiments point to an overall antiinflammatory effect of these algae, indicating that they can reinforce membrane barrier function.     

Dihydroaustrasulfone Alcohol Inhibits PDGF-Induced Proliferation and Migration of Human Aortic Smooth Muscle Cells through Inhibition of the Cell Cycle

Dihydroaustrasulfone alcohol is the synthetic precursor of austrasulfone, which is a marine natural product, isolated from the Taiwanese soft coral Cladiella australis. Dihydroaustrasulfone alcohol has anti-inflammatory, neuroprotective, antitumor and anti-atherogenic properties. Although dihydroaustrasulfone alcohol has been shown to inhibit neointima formation, its effect on human vascular smooth muscle cells (VSMCs) has not been elucidated. We examined the effects and the mechanisms of action of dihydroaustrasulfone alcohol on proliferation, migration and phenotypic modulation of human aortic smooth muscle cells (HASMCs). Dihydroaustrasulfone alcohol significantly inhibited proliferation, DNA synthesis and migration of HASMCs, without inducing cell death. Dihydroaustrasulfone alcohol also inhibited platelet-derived growth factor (PDGF)-induced expression of cyclin-dependent kinases (CDK) 2, CDK4, cyclin D1 and cyclin E. In addition, dihydroaustrasulfone alcohol inhibited PDGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), whereas it had no effect on the phosphorylation of phosphatidylinositol 3-kinase (PI3K)/(Akt). Moreover, treatment with PD98059, a highly selective ERK inhibitor, blocked PDGF-induced upregulation of cyclin D1 and cyclin E and downregulation of p27^kip1. Furthermore, dihydroaustrasulfone alcohol also inhibits VSMC synthetic phenotype formation induced by PDGF. For in vivo studies, dihydroaustrasulfone alcohol decreased smooth muscle cell proliferation in a rat model of restenosis induced by balloon injury. Immunohistochemical staining showed that dihydroaustrasulfone alcohol noticeably decreased the expression of proliferating cell nuclear antigen (PCNA) and altered VSMC phenotype from a synthetic to contractile state. Our findings provide important insights into the mechanisms underlying the vasoprotective actions of dihydroaustrasulfone alcohol and suggest that it may be a useful therapeutic agent for the treatment of vascular occlusive disease.     

Xyloketal B Suppresses Glioblastoma Cell Proliferation and Migration in Vitro through Inhibiting TRPM7-Regulated PI3K/Akt and MEK/ERK Signaling Pathways

Glioblastoma, the most common and aggressive type of brain tumors, has devastatingly proliferative and invasive characteristics. The need for finding a novel and specific drug target is urgent as the current approaches have limited therapeutic effects in treating glioblastoma. Xyloketal B is a marine compound obtained from mangrove fungus Xylaria sp. (No. 2508) from the South China Sea, and has displayed antioxidant activity and protective effects on endothelial and neuronal oxidative injuries. In this study, we used a glioblastoma U251 cell line to (1) explore the effects of xyloketal B on cell viability, proliferation, and migration; and (2) investigate the underlying molecular mechanisms and signaling pathways. MTT assay, colony formation, wound healing, western blot, and patch clamp techniques were employed. We found that xyloketal B reduced cell viability, proliferation, and migration of U251 cells. In addition, xyloketal B decreased p-Akt and p-ERK1/2 protein expressions. Furthermore, xyloketal B blocked TRPM7 currents in HEK-293 cells overexpressing TRPM7. These effects were confirmed by using a TRPM7 inhibitor, carvacrol, in a parallel experiment. Our findings indicate that TRPM7-regulated PI3K/Akt and MEK/ERK signaling is involved in anti-proliferation and migration effects of xyloketal B on U251 cells, providing in vitro evidence for the marine compound xyloketal B to be a potential drug for treating glioblastoma.     

温度对锦鲤疱疹病毒体外培养和致病性的影响

【目的】证实温度对锦鲤疱疹病毒CyHV-3体外增殖和致病性的影响。【方法】选用CyHV-3-EGFP病毒株为研究对象,采用间接免疫荧光染色法测定病毒滴度,使用荧光显微镜观察病毒体外增殖,研究病毒的热稳定性、以及温度对病毒的体外培养和体内致病性的影响。【结果】CyHV-3在4~36℃条件下具有很好的稳定性,温度超过36℃后,病毒活力随着温度升高而降低,50℃时病毒活力完全丧失。感染温度对CyHV-3病毒活力影响不大,高温(37℃)和低温(4℃)时病毒都具有感染能力。但是,孵育温度明显影响病毒活力,温度高于30℃时,CyHV-3的增殖能力减退甚至消失。鲤鱼感染CyHV-3病毒后,低温组(15℃)的死亡率为26.67%,常温组(25℃)的死亡率为73.33%,高温组(30℃)和对照组的死亡率均为0。【结论】证实了CyHV-3病毒在低温下能够增殖并且具有致病性,解释了近年来锦鲤疱疹病毒(KHVD)的暴发及流行不再受限于春秋季节的现象。     

重瓣榆叶梅茎段组织培养体系的建立

以重瓣榆叶梅带腋芽的茎段作为试验材料,研究了不同外植体消毒方式、初代培养基激素配比和生根培养基激素配比对重瓣榆叶梅组织培养的影响。结果表明:1)先用75%酒精处理30 s,再使用0.1%升汞溶液消毒8 min或以2%次氯酸钠溶液消毒12 min,外植体感染率最低;2)茎段初代培养最佳培养基为MS+6-BA2.0 mg/L+NAA 0.05 mg/L,诱导率为89.30%;3)最佳增殖培养基为MS+6-BA 2.0 mg/L+NAA 0.1 mg/L+KT1.0 mg/L,增殖倍数为4.4,且生长状况良好;4)1/2MS+IBA 0.2 mg/L为最佳生根培养基;5)该研究建立了重瓣榆叶梅组培再生体系,为工厂化育苗与大面积推广奠定了基础。     

南高丛蓝莓快繁体系的建立

为建立一套完整、高效的蓝莓快繁体系,以南高丛蓝莓‘南金’的幼嫩枝条为试验材料,取带腋芽的茎段为外植体,分别进行外植体消毒、丛生芽诱导、组培苗继代增殖、组培苗生根和组培苗移栽试验。结果表明:用5%Na Cl O溶液消毒10 min、用0.1%Hg Cl2溶液消毒7 min时外植体的消毒效果最好;蓝莓茎段诱导形成丛生芽的最佳培养基是改良WPM+ZT 4.0 mg/L,增殖倍数达3.86,平均芽高为19.4 mm;组培苗最佳继代增殖培养基是改良WPM+ZT 1.0 mg/L,增殖倍数为1.82,平均株高达44.7 mm;组培苗最佳生根培养基组合是改良1/4 WPM+IBA0.5 mg/L+NAA 1.0 mg/L;组培苗最佳移栽基质是蛭石+草炭土(体积比1∶1),移栽成活率可达96.95%。     

现代商品仔猪传代肾细胞株的培育与应用研究

细胞系是动物病毒分离培养的重要载体。本研究以现代商品化仔猪肾脏为原始材料,拟培育新的细胞系用于动物病毒的分离和培养。利用胰酶消化法和差速贴壁相结合方法,分离纯化仔猪肾上皮细胞,并在体外进行传代培养和筛选。结果显示,试验成功得到一株可以连续传代的细胞株,命名为SDPK-D,且已在体外连续传代90代。SDPK-D细胞株F33和F83代倍增时间分别为40.9和32.7 h,细胞活率分别为97.55%和98.86%,8 h细胞贴壁率分别为91.67%和97.06%。在该细胞株接种猪伪狂犬病病毒（PRV）、猪流感病毒（SIV）、猪流行性腹泻病毒（PEDV）阳性病料均出现明显的细胞病变。本研究首次针对现代商品猪培育出一株可以在体外连续传代的细胞株,并对多种动物病毒敏感,为相关动物病毒的分离培养提供了新的细胞系选择。