黑果枸杞花青素结构差异对其稳定性及细胞抗氧化活性的影响

本文研究了黑果枸杞花青素化合物结构对其稳定性和细胞抗氧化活性的影响。通过半制备型HPLC分离纯化得到了4种黑果枸杞花青素化合物并鉴定结构,利用比色法评价了化合物的稳定性、细胞毒实验以及H₂O₂诱导的SH-SY5Y细胞损伤模型评价了细胞抗氧化活性,并讨论了化学结构对其影响。结果表明反式结构比顺式结构在中性和弱碱性条件下有更好的稳定性,反式结构、糖基的增加以及母核B环上的甲氧基取代均可以通过缩短母核与糖链的分子距离从而提高稳定性,糖基的增加能显著地降低损伤细胞中的乳酸脱氢酶水平,且反式结构较顺式结构更有活性优势。综上所述,黑果枸杞花青素的结构影响稳定性和细胞抗氧化活性,但糖基的增加和酰基的反式异构对二者有更积极的意义。     

响应面法对黑果枸杞中花青素测定条件的优化

以黑果枸杞为原料,采用pH示差法,通过响应面法优化花青素含量的测定条件。通过单因素试验和Box-Behnken Design试验研究乙醇体积分数、pH 1.0缓冲液平衡时间、pH 4.5缓冲液平衡时间等3个变量对黑果枸杞中花青素响应值的影响程度。根据响应面的最优化分析,得出最佳条件是：用80%酸性乙醇溶液,超声萃取30min,取花青素提取液在pH 1.0和pH 4.5的缓冲液中各平衡80min,在最适波长处测定。通过试验验证,采用pH示差法,通过响应面法优化得到的黑果枸杞中花青素含量的测定条件,可靠准确、具有实用价值。     

黑果枸杞的组织培养

1植物名称黑果枸杞(Lycium ruthenicum Murr.). 2材料类别带腋芽的嫩茎段. 3培养条件诱导分化培养基:(1)MS 6-BA 1.0mg·L-1(单位下同) NAA 0.2,(2)MS 6-BA 0.5 NAA0.2;芽增殖培养基:(3)MS 6-BA 0.5 NAA 0.1(4)MS 6-BA 0.5 NA 0.2;生根培养基为:(5)1/2MS NAA 0.1.以上诱导分化及增殖培养基均加2.5%蔗糖和0.8%琼脂,生根培养基的蔗糖和琼脂量减半,pH 5.8～6.0.培养温度为23～25℃,光照时间10～12 h·d-1,光照度为2000 1x.     

‘黑杞一号’与野生黑果枸杞的酚类物质分析

以‘黑杞一号’和野生黑果枸杞为研究对象,对其总酚、总花色苷、总单宁、单体花色苷和非花色苷单体酚类物质进行测定分析。结果表明:(1)与野生黑果枸杞相比,‘黑杞一号’的总酚、总花色苷、总单宁含量分别高出14 250、390和3 330μg/g。(2)‘黑杞一号’和野生黑果枸杞中均检测出4种单体花色苷,包括双葡萄糖苷1种、咖啡酰化葡萄糖苷2种和香豆酰化葡萄糖苷1种,且‘黑杞一号’的4种单体花色苷均显著高于野生黑果枸杞,其中单体花色苷总量比野生黑果枸杞高116.88%(5 672.8μg/g),二甲花翠素咖啡酰化葡萄糖苷含量为野生黑果枸杞的14.93倍。(3)‘黑杞一号’和野生黑果枸杞中均检测到29种非花色苷单体酚,包括黄烷醇类7种、羟基苯甲酸类4种,黄酮醇类18种,并以原儿茶酸含量最高,但‘黑杞一号’非花色苷单体酚总量较野生黑果枸杞低51.42%(2.71μg/g)。     

三氧化二砷通过诱导自噬抑制肝癌细胞增殖和迁移

目的 研究三氧化二砷(As2O3)对肝癌细胞恶性生物学行为的影响并探讨自噬在其中的作用.方法 用0.1μmol/L As2O3处理人肝癌细胞SMMC-7721和HepG2细胞,建立稳定的慢性As2O3诱导细胞系.平板克隆实验、软琼脂集落形成实验和Transwell实验分别用于检测上述慢性As2O3诱导肝癌细胞及其对照细...     

干旱胁迫对黑果枸杞幼苗光合特性的影响

以当年生黑果枸杞幼苗为试验材料,通过称重控水的方法设置对照(土壤含水量为32.96%~35.35%)、轻度干旱胁迫(土壤含水量为21.18%~22.32%)、中度干旱胁迫(土壤含水量为12.20%~13.82%)和重度干旱胁迫(土壤含水量为7.89%~8.73%)4个水分梯度,研究了干旱胁迫对黑果枸杞叶片光合色素、光合特性、叶绿素荧光特性的影响,以揭示黑果枸杞对干旱胁迫的适应能力和适应机制。结果显示:(1)随着干旱胁迫强度的增加,黑果枸杞幼苗叶片叶绿素含量、类胡萝卜素含量均呈显著下降趋势。(2)黑果枸杞幼苗叶片净光合速率(P_n)、蒸腾速率(T_r)、气孔导度(G_s)在中度和重度干旱胁迫下显著下降;其胞间CO_2浓度(C_i)、水分利用效率(WUE)随干旱胁迫强度的增加而逐渐增加,而气孔限制值(L_s)随干旱胁迫强度的增加而逐渐降低。(3)随着土壤含水量的降低,黑果枸杞幼苗叶片初始荧光(F_0)和非光化学猝灭系数(q_N)逐渐增加,而其最大荧光(F_m)、PSⅡ最大光化学效率(F_v/F_m)、实际光化学效率(Ф_(PSⅡ))和光化学猝灭系数(q_P)均逐渐降低。研究表明,在干旱胁迫条件下,黑果枸杞叶片过多的能量以热的形式被耗散,反应中心开放程度降低,从而避免PSⅡ反应中心受到损伤,表现出一定的耐旱性;黑果枸杞生长所允许的最大土壤水分亏缺为7.89%,维持黑果枸杞具有较高的WUE和P_n的土壤水分阈值为12.20%~13.82%。     

CXCL1 在原发性肝癌中的表达及对人肝癌HepG2细胞增殖的影响

目的:探究CXCL1在原发性肝癌中的表达及对人肝癌HepG2细胞增殖的影响。方法:应用免疫组织化学技术(SP二步法)检测48例原发性肝癌组织、13正常肝脏组织中CXCL1表达情况;通过CCK8试剂盒检测CXCL1对HepG2细胞增殖能力的影响。结果:CXCL1在77.1%的原发性肝癌组织中表达,同时CXCL1在原发性肝癌组织中的表达高于正常肝脏组织,差异有统计学意义(P0.01);不同浓度CXCL1处理人肝癌HepG2细胞后,人肝癌HepG2细胞增殖能力明显增强,并且在一定范围内存在明显的剂量效应。结论:CXCL1在原发性肝癌中表达上调;CXCL1能够促进人肝癌HepG2细胞增殖。     

黑果枸杞中十三种元素含量的测定

采用原子吸收法测定了黑果枸杞中钙、镁、铜、锌、锰、铁、铅、镍、镉、钴、铬、钾、钠等元素的含量 ,为对其深入研究提供了科学资料。结果表明 :黑果枸杞中钙、镁、铜、锌、铁等元素的含量远高于宁夏枸杞中相应各元素的平均含量 ,钾、锰含量远低于其平均值 ,钠含量与其相当。钴含量远高于绿叶蔬菜的含量 ,铬含量较牛奶中含量低。镍含量比谷物、腌肉、蔬菜中的高。镉、铅含量较普通植物中的略低 ,不构成污染。     

黑果枸杞的花部结构及繁育系统特征

以宁夏、青海野生分布的黑果枸杞硬枝扦插苗为试验材料,对其开花动态与花部形态特征进行观察,并运用TTC法、联苯胺 过氧化氢法、P/O、OCI和套袋试验等方法针对黑果枸杞花部结构及繁育系统进行研究。结果表明：黑果枸杞5～9月开花,单花持续期2～3 d;黑果枸杞花粉活力在花药开裂时处于最强的状态,达到93.02%,15d后,为2.97%;开花当日黑果枸杞柱头都具有可授性,在散粉后0～36 h内,为传粉受精的最佳时间;杂交指数OCI为3或4,P/O（花粉量与胚珠比）为8 750～10 652,结合坐果率判断黑果枸杞不存在无融合生殖现象,部分自交亲和,繁育系统属于异交,需要传粉者。黑果枸杞的繁育系统以异交为主,但其仍保留着一定的自交花部综合特征。     

Sirt1促进肝癌细胞增殖和侵袭活性的分子机制

目的:探讨Sirt1基因在肝癌组织和癌旁组织中表达差异,进一步检测其对肝癌细胞增殖和侵袭活性的调控.方法:收集30例肝癌手术患者的病变组织和癌旁组织,通过Real time-PCR检测Sirt1基因的表达差异,并对其中6例组织通过western blot验证.在转染Sirt1基因或干扰掉该基因后,采用MTT的方法检测HepG2细胞的增殖活性,通过Transwell小室的方法检测HepG2细胞的侵袭活性.结果:Real time-PCR检测发现Sirt1 mRNA在肝癌组织中高表达,同样,Western blot检测也发现Sirt1在肝癌组织中高表达,而在癌旁组织中表达较低.过表达Sirt1导致HepG2细胞过度增殖,侵袭能力增加;相反,敲除该基因,细胞增殖和侵袭活性被抑制.结论:Sirt1在肝癌组织中高表达并且介导肝癌细胞增殖和侵袭活性.该基因在肝癌组织中的过量表达有助于肝癌的临床诊断,同时Sirt1在肝癌的恶性肿瘤生物活性中发挥着重要的作用,因此,Sirt1是一个潜在的治疗肝癌的药物作用靶点,为开发新的抗肿瘤药物提供了新的治疗靶点.     

黑果枸杞色素的提取和精制工艺研究

本文采用正交实验法对黑果枸杞色素的提取和精制工艺进行了研究。结果表明,黑果枸杞色素的最佳提取条件为:以pH 3.0的80%乙醇作浸提剂,提取温度50℃,提取时间3 h,固液配比1:40;用X-5大孔吸附树脂对色素进行精制,以树脂柱径高比1:15、流速3 mL/minp、H 3.0、色素液浓度1 g/L为最佳吸附条件,色素的吸附量可达0.03715 g/mL湿树脂体积;而以95%乙醇做洗脱液,在pH 2.0、流速5 mL/min、3倍于柱床体积的洗脱液条件下解吸附效果最佳,色素回收率达到97.78%;制取的色素产品外观呈紫红色,色价为36.7。     

药食植物黑果枸杞内生真菌的分离及其挥发性成分分析

从黑果枸杞中分离得到18株黑果枸杞内生真菌,其中从棘刺分离得到3株,从根部分离得到10株,从茎部分离得到5株。对其内生真菌使用固体发酵技术放大培养后,使用固相微萃取材料对黑果枸杞内生真菌挥发性成分进行固定吸附后,经过气相色谱-质谱联用仪(GC-MS)技术解吸附分析。结果表明,4株黑果枸杞植物内生真菌(KLBMPLR001、KLBMPLR004、KLBMPLR013和KLBMPLR018)中的挥发性成分多属于有机酸、芳香烃、酯类及其少量醇类、酚类和倍半萜化合物。4株菌株有机酸百分含量分别为48.69%、51.26%、47.75%和17.78%;芳香烃类百分含量分别为6.79%、19.23%、8.65%和5.75%;酯类百分含量分别为17.18%、7.37%、5.45%和7.95%。     

四逆散对人肝癌HepG2细胞增殖、凋亡的影响及其机制

目的:探讨含四逆散药液血清对人肝癌HepG2细胞增殖、凋亡的影响及机制。方法:将人肝癌HepG2细胞分为5组,每组3个复孔。实验组细胞用五氟尿嘧啶(5-FU)或不同浓度的含四逆散药液血清处理48 h后,用倒置显微镜观察含四逆散药液血清处理后人肝癌HepG2细胞形态的变化; MTT法检测含四逆散药液血清对HepG2细胞生长的抑制作用;荧光染色和流式细胞术分别分析含四逆散药液血清对HepG2细胞凋亡的影响。Rho123染色法检测线粒体膜电位变化,Western blot检测细胞凋亡相关蛋白的表达。结果:与对照组比较,含四逆散药液血清处理人肝癌HepG2细胞后,细胞数量显著减少(P<0.01),形态发生改变,呈现典型的凋亡细胞形态; G1期细胞数明显增加,而G2期细胞数量显著减少(P<0.05); Bax、Caspase-3、-9和Cyt-c的表达显著升高,而Bcl-2的表达显著降低(P<0.05);随着含四逆散药液血清浓度增大,HepG2细胞线粒体膜电位显著下降(P<0.05)。结论:四逆散可以抑制HepG2细胞增殖,并通过线粒体途径诱导细胞凋亡。     

A natural phenylpropionate derivative from Mirabilis himalaica inhibits cell proliferation and induces apoptosis in HepG2 cells

Bioactivity-guided study led to the isolation of a natural phenylpropionate derivative, (E)-3-(4-hydroxy-2-methoxyphenyl)-propenoic acid 4-hydroxy-3-methoxyphenyl ester from the roots of Mirabilis himalaica. Cellular analysis showed that compound 1 specifically inhibited the cancer cell growth through the S phase arrest. Mechanistically, compound 1 was able to induce the apoptosis in HepG2 cells through mitochondrial apoptosis pathway in which Bcl-2 and p53 were required. Interestingly, the cellular phenotype of compound 1 were shown specifically in cancer cells originated from hepatocellular carcinoma (HepG2) while compromised influence by compound 1 were detected within the normal human liver cells (L-02). Consistently, the in vivo inhibitory effects of compound 1 on tumor growth were validated by the in xenograft administrated with HepG2 cells. Our results provided a novel compound which might serve as a promising candidate and shed light on the therapy of the hepatocellular carcinoma.     

Aqueous two-phase assisted by ultrasound for the extraction of anthocyanins from Lycium ruthenicum Murr.

In this study, an efficient ultrasound-assisted aqueous two-phase extraction method was used for the extraction of anthocyanins from Lycium ruthenicum Murr. An ethanol/ammonium sulfate system was chosen for the aqueous two-phase system due to its fine partitioning and recycling behaviors. Single-factor experiments were conducted to determine the optimized composition of the system, and the response surface methodology was used for the further optimization of the ultrasound-assisted aqueous two-phase extraction. The optimal conditions were as follows: a salt concentration of 20%, an ethanol concentration of 25%, an extraction time of 33.7?min, an extraction temperature of 25°C, a liquid/solid ratio of 50:1 w/w, pH value of 3.98, and an ultrasound power of 600?W. Under the above conditions, the yields of anthocyanins reached 4.71?mg/g dry sample. For the further purification, D-101 resin was used, and the purity of anthocyanins reached 25.3%. In conclusion, ultrasound-assisted aqueous two-phase extraction was an efficient, ecofriendly, and economical method, and it may be a promising technique for extracting bioactive components from plants.     

CYP2E1 enhances ethanol-induced lipid accumulation but impairs autophagy in HepG2 E47 cells

The regulation and function of autophagy and lipid metabolism have recently been reported to be reciprocally related. Macroautophagy mediates the breakdown of lipids stored in lipid droplets. An inhibition of autophagy leads to the development of a fatty liver. We evaluated the ability of CYP2E1 to modulate the effects of ethanol on lipid accumulation and autophagy in vitro. The E47 HepG2 cell which expresses CYP2E1 was treated with ethanol at 50, 100 and 150 mM for 4 or 5 days. Ethanol-induced lipid accumulation and an increase of triglycerides (TG) in E47 cells to a greater extent than in control C34 cells which do not express CYP2E1. In contrast, autophagy (LC3 II/LC3 I ratio) was significantly induced by ethanol in C34 cells to a greater extent than in E47 cells. P62 was significantly increased in E47 cells after ethanol treatment. Thus, there is a reciprocal relationship between the effects of ethanol on lipid accumulation and autophagy in the CYP2E1-expressing cells. Inhibition of autophagy by 3-methyladenine (3MA), increased lipid accumulation and TG levels in C34 cells which display elevated autophagy, but enhanced lipid accumulation and TG level to a lesser extent in E47 cells which displayed lower autophagy. Ethanol induced CYP2E1 activity and oxidative stress in E47 cells compared with C34 cells. These experiments suggest that the expression of CYP2E1 may impair autophagy formation which contributes to lipid accumulation in the liver. We hypothesize that CYP2E1-induced oxidative stress promotes the accumulation of lipid droplets by ethanol and this may be responsible for the suppression of autophagy in the liver.     

Nucleostemin siRNA对肝癌细胞HepG2增殖的影响

Nucleostemin(NS)作为核仁蛋白,在神经干细胞、胚胎干细胞以及某些肿瘤细胞中均高表达,在多种肿瘤细胞增殖和凋亡调控中具有重要作用.本文通过瞬时转染NS siRNA降低NS的表达,以探究NS对HepG2细胞增殖和凋亡的影响.结果显示,下调NS表达使HepG2细胞增殖加快,G₁期细胞减少,S期及G₂/M期细胞增加,凋亡减少. 激光共聚焦实验表明,NS与S期激酶相关蛋白2 (S-phase kinase associated protein 2,Skp2)在HepG2细胞中存在共定位现象; Co-IP实验证明,NS与Skp2能相互作用|NS下调后,Skp2出核仁的数量增加,p27和p53表达降低. 总之,下调NS可促进HepG2细胞中Skp2从核仁逸出,p27降解增强,同时p53表达下降,或由此促进HepG2细胞增殖,抑制其凋亡.     

Effect of overexpression and nuclear translocation of constitutively active PKB-alpha on cellular survival and proliferation in HepG2 cells

Protein kinase B (Akt/PKB) is a key component in the PI 3-kinase mediated cell survival pathway and has oncogenic transformation potential. Although the over-expression of PKB-alpha can prevent cell death following growth factor withdrawal, the long-term effects of stable over-expression of PKB-alpha on cell survival in the absence of growth factors remain to be resolved. In the present study, we generated HepG2 cells with stable expression of active PKB-alpha and compared its characteristics with HepG2 cells. Basal as well as insulin-stimulated levels of Ser(473) and Thr(308) phosphorylation in PKB-alpha transfected HepG2 cells were much higher than HepG2 cells. Constitutive expression of active PKB-alpha enabled HepG2 cells to survive up to 96 h without serum in growth media while HepG2 cells fail to survive after 48 h of serum withdrawal. A strong positive correlation (R(2) = 0.71) between cell proliferation and phosphorylated form of PKB-alpha at Thr(308) was observed along with higher levels of phosphorylated 3'-phosphoinositide-dependent kinase-1 (PDK-1). HepG2 cells with constitutive expression of active PKB-alpha also showed higher levels of phosphorylated p65 subunit of nuclear factor-kappaB (NFkappaB) in comparison with HepG2 cells. Predominant nuclear localization of phosphorylated PKB-alpha was observed in stably transfected HepG2 cells. These results indicate that constitutive expression of active PKB-alpha renders HepG2 cells independent of serum based growth factors for survival and proliferation.     

Role of undecan-2-one on ethanol-induced apoptosis in HepG2 cells

Based on the reduced expression of ethanol-oxidizing enzymes in human hepatocellular carcinoma (HepG2) cells, we analyzed the role of nonoxidative metabolites in ethanol-induced apoptosis in HepG2 cells. For this purpose, an analysis of volatile metabolites of ethanol using ion-mobility spectrometry and gas chromatography–mass spectrometry was performed. HepG2 cells exposed to 1 mmol/L ethanol exhibited significant synthesis of undecan-2-one compared to untreated cells. Undecan-2-one is a fatty acid ethyl ester metabolite synthesized through a nonoxidative pathway. Undecan-2-one had a dose-dependent cytotoxic effect on HepG2 cells as shown by release of lactate dehydrogenase (LDH). The most notable finding of this study was the potentiation of ethanol-induced apoptosis demonstrated by an increased apoptotic rate induced by undecan-2-one in ethanol-treated HepG2 cells. The data presented in this study contribute to the better understanding of the molecular mechanisms of ethanol exposure at low concentration in HepG2 cells, a human hepatocellular carcinoma-derived cell line.