羊肚菌胞外多糖提取工艺研究

[目的]探究从羊肚菌发酵液中提取胞外多糖的最佳工艺。[方法]实验原料为羊肚菌发酵液,采用单因素实验的方法探究在不同醇沉浓度、醇沉温度、醇沉时间、醇沉p H的条件下,运用正交实验分析从羊肚菌发酵液中提取多糖的最佳工艺条件;根据实验结果,提取7 d中羊肚菌胞外多糖,分别绘制多糖变化曲线和菌丝体生物量曲线。[结果]通过对羊肚菌胞外多糖提取的研究得到最佳工艺条件为:醇沉浓度95%,醇沉温度-25℃,醇沉时间24 h,醇沉p H 7。[结论]利用优化后的实验条件得出,羊肚菌胞外多糖含量最高为0. 874 g/L,在一定程度上为羊肚菌胞外多糖的研究提供了依据,具有十分重要的意义。     

黄芪多糖和葡萄籽提取物对衰老模型小鼠负重游泳时间和血常规参数的影响

比较日粮中单独添加或同时添加黄芪多糖、葡萄籽提取物对衰老模型小鼠负重游泳时间和血常规参数的影响。采用在日粮中添加0.5%半乳糖构建衰老小鼠动物模型,并将衰老模型小鼠分为模型对照组、黄芪多糖处理组、葡萄籽提取物处理组、黄芪多糖+葡萄籽提取物处理组,每组10只。黄芪多糖和葡萄籽提取物的添加量均为0.1%。试验第42 d时进行负重游泳时间测定,第43 d时眼眶采血,检测血常规参数。结果显示与衰老模型组相比,同时添加黄芪多糖和葡萄籽提取物,可显著提高小鼠的负重游泳时间,显著降低血液粒细胞数和红细胞平均血红蛋白含量;分别单独添加黄芪多糖和葡萄籽提取物均可显著降低红细胞体积分布宽度、血红蛋白浓度和红细胞平均血红蛋白含量。说明黄芪多糖和葡萄籽提取物同时添加对衰老模型小鼠的负重游泳时间有较好延长作用,单独或同时添加黄芪多糖和葡萄籽提取物对于衰老模型小鼠部分血常规参数有一定影响。     

灵芝属多糖对肾上腺酮诱导神经细胞凋亡的保护作用

[目的]筛选和评价灵芝属多糖对肾上腺酮(CORT)诱导神经损伤的保护作用,为灵芝在抗抑郁功能方面的开发奠定基础。[方法]以CORT诱发神经细胞发生损伤后给予灵芝属多糖提取物,分别通过MTT法、乳酸脱氢酶(LDH)胞外释放法、总DNA定量分析法以及荧光标记法确定细胞毒性以及对损伤细胞的保护作用。[结果]结果显示灵芝属多糖在低作用浓度下对PC12细胞不具有毒性,在100μg/mL的作用浓度下灵芝孢子粉多糖(PGL)可以显著提高损伤细胞存活率的比例为22%,减少LDH的释放比例为11.08%,增加胞内总DNA含量14.65%,是所有待测多糖中作用最佳的。[结论]PGL对细胞具有低毒的特点,显著改善CORT对细胞的损伤作用,具有明显的神经保护作用,可作为抗抑郁药物进行研究和开发。     

β-联蛋白促进大鼠外周血间充质干细胞存活及扩增

本研究旨在探讨β-联蛋白在促进外周血间充质干细胞（PBMSCs）的存活和扩增中的作用。采取SD大鼠外周血,分离培养得到PBMSCs,随机将细胞分3组：未处理组（CON）、β-联蛋白激动剂组（WAY-262611,WAY)和β-联蛋白抑制剂组（XAV-939,XAV）。体外培养90 d,观察各组细胞增生和凋亡情况。应用Western 印迹和ELISA方法观察细胞衰老标记性蛋白质p16和p21表达情况,以及β-联蛋白信号通路分子表达情况。外周血经过3代培养,可获得纯度较高的PBMSCs,体外培养40 d即发生衰老和凋亡,但WAY-262611可延长细胞存活时间至90 d以上,增加其体外增殖率1.08倍（P<0.05）,明显提高细胞增殖能力（P<0.05）;使WAY组p21和p16蛋白含量较CON组减少72.1%和68.4%（两者均P<0.05）,较XAV组减低92.9%和93.3%（两者均P<0.05）。与CON组比较,WAY组衰老细胞数减少61.4%（P<0.05）,而XAV组则增加53.3%（P<0.05）;WAY组细胞凋亡率减少50.2%（P<0.05）,而XAV组细胞凋亡率增加35.7%（P<0.05）;WAY组β-联蛋白表达水平增加2.2倍（P<0.05）,而XAV组则降低77.7%（P<0.05）。WAY-262611可升高WAY组β-联蛋白磷酸化水平（包括p-Ser552 和p-Ser675）97%和79%（两者均P<0.05）,升高抗凋亡蛋白质Bcl2表达水平1.39倍（P<0.05）,降低凋亡蛋白Bax和胱天蛋白酶3表达67.6%和83.3%（两者均P<0.05）;而XAV-939则降低Bcl2蛋白表达水平69.1%（P<0.05）,升高Bax和胱天蛋白酶3表达水平1.27倍和1.02倍（两者均P<0.05）。β-联蛋白可能是影响MSCs存活和增殖的重要信号途径。这些发现可能有助于提高用于组织工程的PB-MSCs的体外生产。     

短小芽孢杆菌对盐藻SZ-05的生物量和β-胡萝卜素产量的影响

研究了短小芽孢杆菌（Bacillus pumilus）对盐藻空间诱变株系SZ-05（Dunaliella salina SZ-05）的生物量及β-胡萝卜素积累的影响。结果表明,短小芽孢杆菌显著提高了盐藻SZ-05的生物量和β-胡萝卜素的产量,明显降低了培养体系中的溶解氧和胞外多糖的含量。溶解氧的减少,使得藻细胞的光呼吸作用下降,光合作用速率提高,使藻细胞生物量增加。胞外多糖具有抗氧化作用,胞外多糖的减少可能进一步增加了β-胡萝卜素的合成,从而使β-胡萝卜素在胁迫条件下大幅度增加。     

短小芽孢杆菌对盐藻SZ-05的生物量和β-胡萝卜素产量的影响

研究了短小芽孢杆菌(Bacillus pumilus)对盐藻空间诱变株系SZ-05(Dunaliella salina SZ-05)的生物量及β-胡萝卜素积累的影响。结果表明,短小芽孢杆菌显著提高了盐藻SZ-05的生物量和β-胡萝卜素的产量,明显降低了培养体系中的溶解氧和胞外多糖的含量。溶解氧的减少,使得藻细胞的光呼吸作用下降,光合作用速率提高,使藻细胞生物量增加。胞外多糖具有抗氧化作用,胞外多糖的减少可能进一步增加了β-胡萝卜素的合成,从而使β-胡萝卜素在胁迫条件下大幅度增加。     

液体发酵茯苓胞外多糖的体外降糖效果研究

为探究液体发酵茯苓胞外多糖的体外降糖效果,本研究以高胰岛素抵抗HepG2细胞为受试对象,分析了不同浓度的茯苓胞外多糖对葡萄糖消耗、细胞增殖和糖原合成的影响,并考察了茯苓胞外多糖对α-葡萄糖苷酶的抑制活性。研究结果显示,茯苓胞外多糖可显著促进高胰岛素抵抗HepG2细胞的葡萄糖消耗(p0.05),并加速其糖原合成,但对其细胞增殖无抑制活性;此外,茯苓胞外多糖在终浓度高于2.5μg/mL时还可表现出显著的α-葡萄糖苷酶抑制活性,最大抑制率可达29.51%。研究表明,液体发酵茯苓胞外多糖具有非常好的体外降糖效果。     

当归多糖延缓5-氟尿嘧啶所致人骨髓基质细胞衰老

探讨5-氟尿嘧啶(5-fluorouracil,5-FU)对人骨髓基质细胞(human bone marrow stromal cell,h BMSC)的损伤作用机制;当归多糖(angelica sinensis polysaccharides,ASP)对5-氟尿嘧啶损伤h BMSC的保护作用。采用CCK-8法测定人骨髓基质细胞株HS-5对不同浓度5-FU(0μg/mL,12.5μg/mL,25μg/mL,50μg/mL和100μg/mL)的敏感性。流式细胞术分析细胞周期;β-半乳糖苷酶染色检测衰老细胞;DCFH-DA荧光染色流式检测胞内活性氧(reactive oxygen species,ROS)水平;酶学法检测谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)、超氧化物歧化酶(superoxide dismutase,SOD)含量;流式细胞术分析γH2AX表达水平;ELISA检测8-羟基脱氧鸟苷(8-hydroxydeoxyguanosine,8-OHd G)的含量;5-FU 12.5~100μg/mL抑制HS-5增殖,抑制作用具有浓度依赖性和时间依赖性。5-FU组较与对照组相比细胞周期发生G1期阻滞,β-半乳糖苷酶染色阳性率升高;胞内ROS含量显著升高;细胞抗氧化能力降低;γH2AX和8-OHd G表达水平增高。ASP治疗组较5-FU组细胞相比细胞周期阻滞减少;衰老细胞比率降低;胞内ROS含量显著降低;细胞抗氧化能力升高;DNA损伤指标表达水平降低。5-FU可通过增强氧化应激诱发DNA损伤致骨髓基质细胞衰老;ASP对骨髓基质细胞损伤有保护作用,其机制可能与ASP降低损伤后骨髓基质细胞氧化应激减轻DNA损伤从而延缓骨髓基质细胞衰老有关。     

粗柄羊肚菌菌丝体多糖及胞外多糖的提取工艺

为了研究粗柄羊肚菌菌丝体多糖及胞外多糖的最佳提取工艺条件,采用超声波提取菌丝体多糖、菌丝体发酵液浓缩提取胞外多糖的方法。通过单因素试验和L9(34)正交试验设计,探讨二者的最佳提取工艺条件。研究结果表明菌丝体多糖的最佳提取工艺条件:料液比1∶20(g∶m L),超声波处理温度70℃,超声时间20 min,超声提取次数2次;胞外多糖的最佳提取工艺条件:浓缩倍数1∶5,浓缩温度50℃,醇沉浓度95%,p H为6。该项工艺研究得到菌丝体粗多糖平均含量56.761 2 mg/g,胞外多糖平均含量1.275 4 mg/m L,多糖产量稳定,且此试验方法稳定可行。     

羊肚菌液体发酵培养基的优化及发酵液成分分析

通过液体深层发酵获得羊肚菌菌丝体和发酵液为羊肚菌的开发利用提供了新的途径,本文以胞内多糖和胞外多糖为综合指标,对羊肚菌的最佳培养基组成进行了优化,并对羊肚菌发酵液的成分进行了分析。     

Ethanol extract of Magnoliae cortex (EEMC) limits teratoma formation of pluripotent stem cells by selective elimination of undifferentiated cells through the p53-dependent mitochondrial apoptotic pathway

Background: Induced pluripotent stem cells (iPSCs) are regarded as the best potential cell source for cell-based regenerative medicine. To develop a safe and efficient iPSC-based cell therapy, it is very important to avoid possible teratoma formation, which can arise from undifferentiated iPSCs (USCs) remaining among differentiated cell products. Dried bark of Magnolia officinalis (Magnolia cortex, MC) has long been used in traditional medicine to treat gastrointestinal ailments and allergic diseases, and has shown have various pharmacological activities, including anti-bacterial, anti-inflammatory, and anti-cancer effects. However, its effects on iPSCs have not yet been examined.Purpose: In this study, we investigated the selective cytotoxic effects of ethanol extract of MC (EEMC) on undifferentiated iPSCs and elucidated the underlying apoptotic mechanisms in detail. We also investigated the inhibitory effects of EEMC on teratoma formation via in ovo experiments.Results: We found that EEMC greatly reduced cell growth and induced apoptotic cell death in USCs, but not in differentiated or normal cells. EEMC caused G2/M cell cycle arrest, mitochondrial damage, and caspase activation of USCs, accompanied by p53 accumulation. In p53KO human iPSCs, EEMC had no cytotoxicity, reinforcing that EEMC-mediated apoptosis of USCs is p53-dependent. EEMC did not cause DNA damage in iPSC-derived differentiated cells. In ovo teratoma formation assay revealed that EEMC treatment before injection efficiently eliminated USCs and prevented teratoma formation.Conclusions: These results collectively indicate that EEMC has potent anti-teratoma activity, and therefore can be used for the development of safe iPSC-based therapy.     

Augmentation of prostaglandin and thromboxane production in vitro by monocytes exposed to histamine-induced suppressor factor (HSF)

A possible mechanism to explain the suppression of mitogen-induced lymphocyte proliferation in vitro by histamine-stimulated mononuclear cells was investigated. In initial experiments, the inhibitory action of histamine-induced suppressor factor (HSF) on lymphocyte proliferation was documented to be reduced by the addition of indomethacin (1 μg/ml). Moreover, the addition of exogeneous PGE₂ (10^?7-10^?8 M) to mononuclear cell cultures reconstituted HSF activity in the presence of indomethacin. In order to ascertain the nature of the target cell responding to HSF, control and suppressor supernatants were incubated with human lymphocytes or monocytes (5 × 10⁶ cells/ml) for 24 hr. Following incubation, the supernatants were assayed for their content of prostaglandin E₂, F_2α, and thromboxane B₂. Monocytes (but not lymphocytes) incubated with supernatants containing HSF increased their production of prostaglandin E₂, F_2α, and thromboxane B₂ by 169, 53, and 49%, respectively. Suppressor supernatants were generated with histamine or an H-2 agonist (dimaprit) and chromatographed by gel filtration on Sephadex G-100. The elution profiles for the factor(s) inducing suppression of lymphocyte proliferation (25–40,000 daltons) and augmenting PGE₂ production (25,000 daltons) overlapped but were not identical. Collectively, these data suggest that HSF-mediated inhibition of lymphocyte proliferation may occur in part through the augmented production of prostaglandins and/or thromboxane B₂ by human monocytes.     

磷酸三钙涂层镁合金材料的细胞相容性研究

目的:制备磷酸三钙(β-TCP)涂层镁合金材料,评价材料表面的特性及体外的细胞生物适应性。方法 电化学法制备β-TCP涂层镁合金材料(β-TCP-Mg-AI-Zn),观测金属材料表面微观结构特性和能谱分析,小鼠颅骨源成骨细胞与材料直接接触培养,荧光染色观察材料表面细胞生长状况,检测成骨细胞增殖和碱性磷酸酶(ALP)活性。结果 β-TCP涂层Mg-AI-Zn材料表面呈多孔状,材料表面含有镁、钙和磷等元素;成骨细胞与材料直接接触培养24 h及48 h后,材料表面有大量的成骨细胞粘附、伸展、汇合;与Mg-AI-Zn材料比较,β-TCP-Mg-AI-Zn材料明显地促进细胞增殖、显著地增加成骨细胞中ALP活性 (P＜0.05)。结论 β-TCP涂层改善了Mg-AI-Zn镁合金材料表面特性及体外的细胞相容性,有望成为新一代可降解医用金属材料。     

Inhibition of cell metabolism by a smokeless tobacco extract: tissue and species specificity.

Smokeless tobacco contains a nonnicotine inhibitor of posttranslational modification of collagen (hydroxylation of [3H]proline) by cultured chick embryo tibias and osteoblasts. This study was undertaken to determine whether a methanol extract of smokeless tobacco (STE) containing the inhibitor has similar effects on collagen-producing cells and tissues other than bone. Its effects on DNA synthesis and cell proliferation (incorporation of [3H]thymidine) were also determined. Frontal bone, aorta, and cartilage were incubated for 2 days in medium containing STE. Glycolysis (lactate production) was stimulated by 80% in cartilage, but was not affected in the other tissues; medium alkaline phosphatase activity was unaffected. In frontal bone and cartilage, [3H] hydroxyproline content was decreased 88% and 57%, respectively, and [3H]proline content was decreased 68% and 37%, respectively; neither was affected in the aorta. Confluent cultures of collagen-producing mouse fibroblasts or primary osteoblasts obtained from chick embryo calvarias were incubated for 2 days in medium containing increasing concentrations of STE. Glycolysis and DNA synthesis were not affected. Cell proliferation was unaffected in fibroblasts, but was inhibited (34%) at the highest STE concentration in osteoblasts. AIPase activity was not detectable in fibroblast medium, but was decreased up to 72% in osteoblast medium. Inhibition of collagen synthesis by STE was concentration related in both cell types. At the highest concentration, [3H] hydroxyproline and [3H]proline contents in the cell layers were decreased to the following respective values: fibroblasts 56% and 45% and osteoblasts 50% and 29%, respectively. When incubation with STE was discontinued for 1 day, recovery did not occur. These findings suggest that inhibition of collagen synthesis by STE is not specific for bone, that collagen-producing cells are directly affected, and that recovery is not immediate. This inhibitor could contribute to the periodontal disease often seen in users of smokeless tobacco. Its identification and removal would produce a safer product.     

Growth Stimulation of Human Skin Fibroblasts by Elastin-Derived Peptides

Elastin-derived peptides (kappa-elastin: KE, mean molecular mass: 75 kDa), either coated onto plastic dishes or added to culture media (0.26 to 1.33 nM) stimulated the growth of human skin fibroblasts (HSF) strains obtained from different donors and tested at different cell passages (4 to 12). Coated 44.4 μg/cm²insoluble elastin (iE) exhibited the same action; coated iE or KE significantly modifies the HSF morphology: after 5-6 days of culture, HSF are more elongated, and at preconfluence state, formation of HSF clusters surrounding iE were observed. Increased ³H thymidine incorporation and proliferative effect of HSF by KE (1.3 to 2.2 fold as compared to control cells) was observed after a lag phase period which raised with initial HSF density. Optimal proliferative effect was obtained at KE 8.5 10^-10M, a value close to the dissociation constant (k_D= 2.7 10^-10M) of KE to HSF. Valine-glycine-valine-alanine-proline-glycine (VGVAPG), but not valine-glycine-valine (VGV) or Valine-glycine-valine-valine-glycine-alanine (VGWGA) also significantly stimulated, optimally at 7.0 10^-10M, HSF proliferation. It was concluded that the stimulatory influence of elastin derived peptides on HSF proliferation was mediated through a binding to plasmalemmal receptor of HSF.