忍冬木层孔菌多糖的提取工艺优化及其体外抗氧化活性研究

忍冬木层孔菌(Phellinus lonicerinus(Bond.) Bond.et Sing)为湖北等地习用的桑黄品种,也是土家族常用药,多糖是其主要活性成分。为了研究忍冬木层孔菌子实体多糖(Phellinus lonicerinus polysaccharide, PLP)的提取工艺及其体外抗氧化活性,采用冷凝回流提取法在单因素实验结果的基础上,进行Box-Behnken响应面分析,确定PLP的最佳提取工艺,并通过测定忍冬木层孔菌多糖(PLP30、PLP60、PLP85、PLPA1和PLPA2)的还原能力、对DPPH、ABTS、羟基自由基的清除能力、对酪氨酸酶活性的抑制能力。结果显示PLP最佳提取工艺的参数为：料液比1∶20 g/mL、提取时间120 min、提取温度100℃,此条件下PLP提取率为(3.16±0.05)%;五种多糖对DPPH、ABTS和羟基自由基均具有较强的清除作用,且具有较强的还原力,其对ABTS自由基清除作用和还原力均与浓度呈量效关系。表明忍冬木层孔菌子实体多糖具有良好的体外抗氧化和一定的酪氨酸酶活性抑制能力。     

鲍姆木层孔菌子实体中化合物的结构鉴定及其抗肿瘤活性

鲍姆木层孔菌(Phellinus baumii)俗称桑黄菇,桑耳,是近年来开发的一种多年生药用真菌。已经有研究证实,桑黄具有降血糖,抗氧化,抗肿瘤等生物活性。该研究从化学成分的分离纯化和结构鉴定入手,寻找鲍姆木层孔菌子实体中具有抗肿瘤活性的化合物,为桑黄保健品的开发提供研究基础。采用色谱柱层析法,从鲍姆木层孔菌子实体中分离得到6个化合物,通过波谱分析,分别鉴定为Ergosta-7,22-diene-3β-ylpentadecanoate,Stellasterol,Ganoderol B,Ergos-t6,22-diene-3β,5α,8α-triol,Ganoderic acid DM,Inoscavin A。体外试验结果表明:化合物GanoderolB和Inoscavin A对肿瘤细胞HepG2的增殖均有抑制作用。其中,化合物Inoscavin A的抑制作用较强,其抑制肿瘤细胞增殖的IC50值为72.3μg/mL(156.4μmol/L)。     

斜生褐孔菌多糖诱导人肝癌HepG-2细胞凋亡的研究

研究了斜生褐孔菌多糖对人类肝癌HepG-2细胞凋亡的诱导作用。采用噻唑蓝法(MTT法)观察了斜生褐孔菌多糖对HepG-2细胞生长的影响,用透射电镜观察了细胞形态,用DNA Ladder检测了细胞凋亡,用流式细胞仪检测了细胞凋亡率;同时采用逆转录-聚合酶链反应法(RT-PCR法)研究了不同浓度斜生褐孔菌多糖作用后HepG-2细胞中Bax和Bcl-2基因mRNA转录水平的变化。结果表明斜生褐孔菌多糖能抑制HepG-2细胞增殖,并呈时间剂量依赖关系;电镜观察、DNALadder和流式细胞仪检测均证实了斜生褐孔菌多糖能够诱导HepG-2细胞凋亡;经斜生褐孔菌多糖处理后,HepG-2细胞中Bax基因mRNA转录水平增强,而Bcl-2基因mRNA无明显变化。证明了斜生褐孔菌多糖具有抑制HepG-2细胞生长及诱导HepG-2细胞凋亡的作用,这可能与调节Bcl-2和Bax基因表达水平有关。     

三种木层孔菌子实体不同溶剂提取物抗氧化活性的研究

以抗坏血酸为阳性对照,分别对桑木层孔菌Phellinus mori、鲍姆木层孔菌Phellinus baumii和瓦宁木层孔菌Phellinus vaninii野生子实体甲醇提取物(ME)、氯仿提取物(CE)、乙酸乙酯提取物(EAE)、正丁醇提取物(BE)和热水提取物(WE)的体外清除DPPH(1,1-二苯基-2-三硝基苯肼)自由基、羟自由基和总抗氧化能力进行了检测。结果表明,3种木层孔菌的不同溶剂提取物都具有体外抗氧化活性。3种菌EAE清除DPPH自由基能力较强,其中瓦宁木层孔菌EAE清除DPPH自由基能力最强,IC50为10.65μg/mL。3种木层孔菌热水浸提物清除羟自由基的能力较强,鲍姆木层孔菌WE的清除率最高,1mg/mL时达到73.52%。另外,3种木层孔菌不同提取物总抗氧化能力和清除DPPH自由基能力基本相同,瓦宁木层孔菌BE的总抗氧化能力最强,1mg/mL时达到了167.7U/mL。3种木层孔菌相比,瓦宁木层孔菌不同提取物的体外抗氧化能力最强,其次是鲍姆木层孔菌,桑木层孔菌的体外抗氧化能力最弱。     

乙型肝炎病毒对HepG2细胞侵袭的影响

目的研究HBV全长对HepG2细胞侵袭相关基因表达及活性的影响,探讨HBV在整体水平对HepG2细胞侵袭的影响。方法采用定量PCR分析HBV对HepG2细胞MMP2、9和TIMP1-4基因转录的影响;通过明胶酶谱及反相明胶酶谱检测MMP2、MMP9及TIMPs的活性;应用体外侵袭小室法检测细胞的侵袭能力。结果HBV的复制可以促进HepG2细胞MMP2、MMP9、TIMP1和TIMP3基因的转录,抑制TIMP4基因转录,增强HepG2细胞MMP2、MMP9的活性并增强细胞中TIMP1、TIMP3功能,HBV稳定复制的细胞具有更强的体外侵袭能力。结论HBV可影响HepG2细胞MMPs和TIMPs的基因转录、表达及功能,促进HepG2细胞的体外侵袭,这可能与HBV相关的HCC侵袭转移密切相关。     

裂蹄木层孔菌子实体水提物诱导HepG2细胞凋亡的初步研究

研究裂蹄木层孔菌子实体水提物(WEPL)对人类克隆肝癌细胞系HepG2生长的作用。用裂蹄木层孔菌子实体水提物处理HepG2细胞后,噻唑蓝法(MTT法)可见浓度和时间依赖性抑制细胞增殖;电镜下观察凋亡小体的出现,流式细胞仪技术显示Annexin-Ⅴ染色呈阳性,都证明了HepG2细胞发生了凋亡。RT-PCR和Western Blot分析证实WEPL刺激Bax表达量上调、Bcl-2表达量下调进而诱导了细胞凋亡。结果表明WEPL诱发的克隆人类肝癌细胞系HepG2的细胞凋亡可能是通过上调Bax、下调Bcl-2活性来实现的。     

“桑黄”的本草考证

对“桑黄”的古代、现代名称及其药用功效等方面做了全面考证.“桑黄”最早在汉代的《神农本草经》中记载为“桑耳”,在唐代甄权的《药性论》中才首次出现“桑黄”两字.认为中国“桑黄”,在分类上属于真菌界Fungi、担子菌门Basidiomycota、绣革孔菌目Hymenochaetales、绣革孔菌科Hymenochaetaceae,主要包括来自Phellinus、Inonotus及Fomitiporia等属的具有显著抗肿瘤活性的木腐菌子实体,包括火木层孔菌Phellinus igniarius、瓦尼木层孔菌Phellinus vaninii、鲍姆木层孔菌Phellinus baumii、椭圆嗜蓝孢孔菌Fomitiporia ellipsoidea及粗毛纤孔菌Inonotus hispidus等的子实体,并具有明显的地域特色.     

姜黄素对肝癌细胞HepG2 的抗癌作用及P21WAF1/CIP1 表达的影响

目的:探讨姜黄素对肝癌HepG2细胞抗癌作用及相关周期蛋白依赖激酶抑制因子P21WAF1/CIP1表达的影响.方法:体外培养肝癌HepG2细胞,用MTT法检测姜黄素对HepG2细胞的抑制作用,以RT-PCR方法检测HepG2细胞中P21WAF1/CIP1mRNA的表达,用免疫细胞化学检测其P21WAF1/CIP1蛋白的表达.结果:姜黄素呈时间剂量性抑制HepG2细胞的生长,并显著上调HepG2细胞中P21WAF1/CIP1mRNA和蛋白的表达.结论:姜黄素能抑制HepG2细胞的生长,并上调其中P21WAF1/CIP1的表达.     

华蟾素对子宫内膜癌HEC-1-B细胞株凋亡和增殖的影响

目的：探讨华蟾素对体外培养子宫内膜癌HEC-1-B细胞凋亡、增殖以及侵袭能力的影响。方法：体外培养HEC-1-B细胞,经不同浓度华蟾素（0．2mg／ml、2mg／ml和20mg／m1）干预24h后,采用MTT法观察细胞生长情况,流式细胞术分析细胞凋亡,Transwell小室检测细胞体外侵袭能力。结果：华蟾素能有效抑制HEC．1．B细胞生长,诱导细胞凋亡。未经华蟾素处理的细胞凋亡率仅为2．5％,经0．2mg／ml、2mg／ml、20mg／ml的华蟾素作用24h后,细胞凋亡率分别为7．4％、44．3％和78．5％。趋势卡方检验x^2=165．4983,P〈0．0001。HEC-1-B细胞经华蟾素作用后,细胞侵袭能力降低。在高浓度时,华蟾素有可能存在细胞毒作用。结论：华蟾素能通过诱导HEC-1-B细胞凋亡,从而抑制HEC-1-B细胞生长,并且降低细胞的侵袭能力。     

鲍姆木层孔菌（桑黄）脂溶性提取物对PC12神经元细胞衰老的保护作用

鲍姆木层孔菌（桑黄）Phellinus baumii脂溶性提取物对叠氮钠诱导PC12神经元细胞衰老的保护作用。用叠氮钠作用于体外培养的PC12细胞,并用该菌脂溶性提取物对其进行保护,分别用MTT法、流式细胞仪法、细胞核染色法和β-半乳糖苷酶检测试剂盒检测细胞活力、早期凋亡率、晚期凋亡率和β-半乳糖苷酶阳性细胞率变化,筛选出具有延缓细胞衰老的有效部位和有效作用浓度。通过细胞活力检测发现正丁醇萃取的活性部位可以有效保护叠氮钠诱导的细胞活力下降,其有效工作浓度为200mg/L。流式细胞仪和细胞核染色测定结果均表     

Polysaccharides from Phellinus linteus inhibit cell growth and invasion and induce apoptosis in HepG2 human hepatocellular carcinoma cells

It has been demonstrated that the medicinal mushroom Phellinus linteus (PL), which consists mainly of polysaccharides, possesses antitumor and immunomodulatory properties in vivo and in vitro. The mechanism, however, by which PL inhibits growth and invasive behavior of HepG2 cells remains poorly understood. Here we demonstrated that PL inhibited proliferation and colony formation of HepG2 and that the growth inhibition of HepG2 cells was mediated by S-phase cell cycle arrest. PL also markedly inhibited cancer cell adhesion and invasion of the extracellular matrix. Additionally, we demonstrated that PL-induced apoptosis was associated with a reduction in B-cell lymphoma 2 levels and an increase in the release of cytochrome c. These results suggest that PL exerts a direct antitumor effect by initiating apoptosis and cell cycle blockade in HepG2 cells.     

Sulfation of a polysaccharide obtained from Phellinus ribis and potential biological activities of the sulfated derivatives

The paper reports the preparation, characterization and potential biological activities of a chemically sulfated polysaccharide isolated from Phellinus ribis. Four sulfated derivatives (PRP-SI–IV) with variable degrees of substitution were obtained by the chlorosulfonic acid method, without degradation of the polysaccharide (PRP). The sulfate groups were not regularly distributed along the polysaccharide chain with a multiple substitution pattern as determined by ¹³C NMR. The sulfated derivatives except for PRP-SI showed significant inhibition effects on HepG2 cells in comparison with the native non-sulfated polysaccharide (PRP). All sulfated derivatives could block new angiogenic vessel formation in zebrafish assay, however, the effects were less than PRP.     

Lipopolysaccharide promotes adhesion and invasion of hepatoma cell lines HepG2 and HepG2.2.15

Inflammation and infection have been linked to the bionomics of many cancers, including hepatocellular carcinoma (HCC). Some bacteria, such as Helicobacter pylori, have been found in pathological specimens from patients with HCC. However, little is known about the direct effects of bacteria or their components on hepatoma cells. We analyzed the in vitro proliferation, adhesion and invasion responses of the tumor cell lines HepG2 and HepG2.2.15 to lipopolysaccharide (LPS), a cell wall constituent of Gram-negative bacteria. HepG2 and HepG2.2.15 cells show increased proliferation in response to LPS. Their invasion and adhesion abilities were also increased in response to LPS, which may be related to increased gene expression of interleukin-8 and transforming growth factor-β1. We infer that bacteria may be ignored by immune systems and directly promote adhesion and invasion of hepatoma cells through LPS.     

药用真菌桑黄的研究进展

以近年来国内外发表的文献为依据 ,从形态特征、生物学生长特性、主要化学成分、药理学研究等方面综述了文献中称为“桑黄”[包括 :鲍氏针层孔菌 (Phellinusbaumii)、火木针层孔菌 (P .igniarius)、裂蹄针层孔菌 (P .linteus) 3种 ]的药用菌子实体、发酵菌丝体、发酵胞外物的研究进展。结果 :桑黄的人工栽培 (日本、韩国学者采用的室外荫棚段木埋畦栽培法 ) ,固体、液体发酵培养均已获成功。其主要化学成分是子实体、菌丝体多糖以及发酵液中的胞外多糖 ,此外还有黄酮及其衍生物、香豆素类、甾醇类化合物。其子实体、菌丝体及提取物 (主要为多糖 )、胞外多糖在抗发炎、抗氧化、抑制肿瘤生成、增强免疫力、保护肝脏、预防和治疗关节炎等方面有显著的功效。桑黄的各种产品 (包括 :子实体、菌丝体微粉末、提取物浸膏、桑黄茶、桑黄口服液等 )市场需求量很大 ,表明桑黄有广阔的发展前景。     

Retracted:Propofol exerts anticancer activity on hepatocellular carcinoma cells by raising lncRNA DGCR5

Hepatocellular carcinoma is one of the most fatal cancers worldwide. Propofol is an intravenous anesthetic extensively used in clinical. Herein, we tested the anticancer activity of propofol on hepatocellular carcinoma, along with the internal molecular mechanism related to lncRNA DiGeorge syndrome critical region gene 5 (DGCR5). Followed by propofol stimulation, hepatocellular carcinoma Huh-7 and HepG2 cell viability, proliferation, migration, invasion, and apoptosis were tested, respectively. Then, DGCR5 expression levels in hepatocellular carcinoma tissues and cells were measured. sh-DGCR5 was transfected to silence DGCR5 expression. Subsequently, the influence of DGCR5 silence on propofol caused Huh-7 and HepG2 cell viability loss, proliferation inhibition, migration and invasion suppression, apoptosis induction, as well as Raf1/ERK1/2 and Wnt/β-catenin pathways inactivation were assessed, respectively. We discovered that propofol declined Huh-7 and HepG2 cell viability, proliferation, migration and invasion, but increased cell apoptosis. DGCR5 had a relatively lower expression level in hepatocellular carcinoma tissues and cells. Propofol elevated DGCR5 expression in Huh-7 and HepG2 cells. Increased expression of DGCR5 was connected with the anticancer activity of propofol on Huh-7 and HepG2 cells. Besides, propofol repressed Raf1/ERK1/2 and Wnt/β-catenin pathways through elevating DGCR5 expression. In conclusion, the anticancer activity of propofol on hepatocellular carcinoma was verified in this study. Propofol repressed hepatocellular carcinoma Huh-7 and HepG2 cell growth and metastasis at least by elevating DGCR5 and hereafter inactivating Raf1/ERK1/2 and Wnt/β-catenin pathways.     

Morphological and rheological properties of the three different species of basidiomycetes Phellinus in submerged cultures

AIMS: The objective of this work was to investigate the morphological and rheological properties in submerged culture of the three different basidiomycetes Phellinus (P. baumii, P. gilvus and P. linteus) that produce pharmacologically important exopolysaccharides (EPS). METHODS AND RESULTS: In flask cultures, pH proved to be a critical factor affecting mycelial growth, morphological change and EPS production. The macroscopic morphologies observed under different pHs in flask cultures were also comparable: i.e. starfish-like pellets with a lesser extent of free mycelium appeared in P. baumii, whereas smooth pellets with higher amounts of free mycelium were observed in P. gilvus and P. linteus. The pelleted fermentations were further characterized in a 5-l stirred-tank fermenter by image analysis with respect to mean diameter, core area and pellet circularity. Phellinus baumii showed the largest pellet size (5.2 mm in diameter), whereas P. linteus had extremely small and spherical pellets. The culture broth of P. baumii and P. gilvus yielded extremely high apparent viscosities, ranging from 5 to 7 Pa s. CONCLUSIONS: Three important species of Phellinus showed significantly different morphological and rheological properties. The morphological variation of the three Phellinus species was closely linked to EPS productivity and the apparent viscosity of the whole broth. SIGNIFICANCE AND IMPACT OF THE STUDY: The morphological change in the three species of Phellinus was a good indicator for identifying cell activity for EPS production. Our finding may be beneficial for further optimization of other fungal fermentation processes for large-scale production of EPS.     

珊瑚树Vibsane型二萜类化合物对人体HepG2细胞增殖的影响及其机制

目的：探讨珊瑚树vibsane型二萜类化合物对肝癌HepG2细胞增殖的影响及其机制,为研发新型天然植物类抗肿瘤药物提供实验依据。方法：采用噻唑蓝比色法及苔盼蓝染色计数法观察珊瑚树vibsane类二萜类化合物对不同肿瘤细胞增殖的影响;应用流式细胞仪检测细胞周期及细胞凋亡,利用Apo-ONE Homogeneous Caspase-3/7试剂盒检测vibsane二萜类化合物1#对HepG2细胞内Caspase-3酶活性的影响。结果：活性筛选发现vibsane型二萜类化合物1#显著抑制人肝癌HepG2细胞增殖,构效分析表明化合物C11位连接侧链的基团修饰影响其细胞增殖抑制活性。此外,HepG2细胞对1#化合物最敏感,1#化合物抑制其增殖具有剂量和时间依赖性。机制研究显示1#化合物诱导HepG2细胞发生明显的细胞周期G0/G1期阻滞,具有时间和剂量效应;同时,较高浓度1#化合物（5-10μmol/L）引起HepG2细胞凋亡明显增加,并剂量依赖性诱导细胞内Caspase3/7激活。结论：珊瑚树vibsane型二萜类化合物能够明显抑制人肝癌HepG2细胞增殖,其可能通过诱导细胞周期阻滞和细胞凋亡发挥抗肿瘤作用。     

Time- and concentration-dependent effects of resveratrol in HL-60 and HepG2 cells

Resveratrol, a phytochemical present in grapes, has been demonstrated to inhibit tumourigenesis in animal models. However, the specific mechanism by which resveratrol exerts its anticarcinogenic effect has yet to be elucidated. In the present study, the inhibitory effects of resveratrol on cell proliferation and apoptosis were evaluated in the human leukaemia cell line HL-60 and the human hepatoma derived cell line HepG2. We found that after a 2 h incubation period, resveratrol inhibited DNA synthesis in a concentration-dependent manner. The IC50 value was 15 microm in both HL-60 and HepG2 cells. When the time of treatment was extended, an increase in IC50 value was observed; for example, at 24 h the IC50 value was 30 microm for HL-60 cells and 60 microm for HepG2 cells. Flow cytometry revealed that cells accumulated in different phases of the cell cycle depending on the resveratrol concentration. Furthermore, an increase in nuclear size and granularity was observed in the G1 and S phases of HL-60 treated and HepG2-treated cells. Apoptosis was also stimulated by resveratrol in a concentration-dependent manner in HL-60 and HepG2 cells. In conclusion, resveratrol inhibits cell proliferation in a concentration- and time-dependent manner by interfering with different stages of the cell cycle. Furthermore, resveratrol treatment causes stimulation of apoptosis as well as an increase in nuclear size and granularity.     

外泌体介导长链非编码RNA H19促进肝癌细胞增殖与转移

外泌体是由细胞分泌的直径为30～150 nm的小囊泡,含有丰富的mRNA、microRNA和长链非编码RNA(lncRNA)。目前,大多数外泌体研究都集中在mRNA和microRNA,而对lncRNA的生物学功能并不十分清楚。研究表明,肿瘤细胞外泌体 lncRNA H19在肿瘤细胞的增殖、迁移和侵袭中发挥了重要作用。本研究将筛选到的lncRNA H19高表达的肝癌细胞HCCLM3,分别收集其高表达lncRNA H19的外泌体和其下调lncRNA H19表达后的外泌体。然后,将收集到的外泌体分别添加到lncRNA H19低表达的肝癌细胞Hep3B和HepG2孵育液中。孵育24 h后,检测其对肿瘤细胞的增殖、迁移和侵袭能力的影响。结果显示,肝癌细胞HCCLM3可分泌大量的外泌体,且能被其他肿瘤细胞大量摄取;与下调lncRNA H19表达的外泌体相比,lncRNA H19高表达的外泌体能显著增强Hep3B和HepG2细胞的增殖、迁移和侵袭能力。而这一作用可通过激活PI3K/AKT/mTOR通路实现。上述结果表明,lncRNA H19高表达的肝癌细胞以外泌体方式,增强邻近肝癌细胞的增殖、迁移和侵袭能力,促进肝癌的发生与发展。