Rapid Propagation of Pteris vittata L.via Tissue Culture

[Objective] The study had developed a means of rapid propagation Pteris vittata L.by tissue culture. The species was a perennial fern belonging to the genus Pteris. [Metbed] The leaf bud of P. vittata collected in field conditions as explantsand the 1/2 MS ＋ 3% sucrose ＋ 0.7% agar as the basic medium were used to screen the medium formula of the phytohormone ratio for callus induction and subculture of P. vittata. [Result] The best medium formula for each step was list below： 1/2 MS ＋ 3% sucrose ＋ 0.7% agar ＋ 0.5 g/L PVP ＋ 0.1 mg/L KT ＋ 0.5 mg/L 2, 4-D for in- ducing the callus from explants; 1/2MS ＋ 3% sucrose ＋ 0.7% agar ＋ 0.5 g/L PVP ＋ 1.0 mg/L KT ＋ 0.01 mg/L 2,4-D for inducing the GGB from callus and the seedlings from GGB. In addition, 1/2 MS ＋ 3% sucrose ＋ 0.7% agar ＋ 0.5 g/L PVP ＋ 0.5 mg/L 2,4-D for the subculture could make the continued proliferation of callus. [Cen- clusioa] This study makes an applicable procedure by the direct use of field materi- als, for propagating P. vittata in a simplified and rapid mode.

Potential of Pteris vittata L. for phytoremediation of sites co-contaminated with cadmium and arsenic: The tolerance and accumulation

Field investigation and greenhouse experiments were conducted to study the tolerance of Pteris vittata L. （Chinese brake） to cadmium （Cd） and its feasibility for remediating sites co-contaminated with Cd and arsenic （As）. The results showed that P. vittata could survive in pot soils spiked with 80 mg/kg of Cd and tolerated as great as 301 mg/kg of total Cd and 26.8 mg/kg of diethyltriaminepenta acetic acid （DTPA）-extractable Cd under field conditions. The highest concentration of Cd in fronds was 186 mg/kg under a total soil concentration of 920 mg As/kg and 98.6 mg Cd/kg in the field, whereas just 2.6 mg/kg under greenhouse conditions. Ecotypes of P. vittata were differentiated in tolerance and accumulation of Cd, and some of them could not only tolerate high concentrations of soil Cd, but also accumulated high concentrations of Cd in their fronds. Arsenic uptake and transportation by P. vittata was not inhibited at lower levels （〈20 mg/kg） of Cd addition. Compared to the treatment without addition of Cd, the frond As concentration was increased by 103.8% at 20 mg Cd/kg, with the highest level of 6434 mg/kg. The results suggested that the Cd-tolerant ecotype of P. vittata extracted effectively As and Cd from the site co-contaminated with Cd and As, and might be used to remediate and revegetate this type of site.

Arsenic uptake and transport of Pteris vittata L.as influenced by phosphate and inorganic arsenic species under sand culture

In order to understand the similarity or difference of inorganic As species uptake and transport related to phosphorus in Ashyperaccumulator, uptake and transport of arsenate （As（V）） and arsenite （As（Ⅲ）） were studied using Pteris vittata L. under sand culture. Higher concentrations of phosphate were found to inhibit accumulation of arsenate and arsenite in the fronds of P. vittata. The reduction in As accumulation was greater in old fronds than in young fronds, and relatively weak in root and rhizome. Moderate increases, from 0.05 to 0.3 mmol/L, in phosphate reduced uptake of As（Ⅲ） more than As（Ⅴ）, while the reverse was observed at high concentrations of phosphate （≥1.0 mmol/L）. Phosphate apparently reduced As transport and the proportion of As accumulated in fronds of P. vittata when As was supplied as As（Ⅴ）. It may in part be due to competition between phosphorus and As（Ⅴ） during transport. In contrast, phosphate had a much smaller effect on As transport when the As was supplied as As（Ⅲ）. Therefore, the results from present experiments indicates that a higher concentration of phosphate suppressed As accumulation and transport in P. vittata, especially in the fronds, when exposed to As（Ⅴ）, but the suppression of phosphate to As transport may be insignificant when P. vittata exposed to As（Ⅲ） under sand culture conditions. The finding will help to understand the interaction of P and As during their uptake process in P. vittata.

Role of trichome of Pteris vittata L.in arsenic hyperaccu-mulation

Environmental scanning electron microscope(ESEM)fitted with an energy disper-sive X-ray microanalyzer(EDX)was used to investigate the surface micromorphology and arse-nic(As)micro-distribution in Chinese brake(Pteris vittata L.).It was found that amounts of trichome,which possessed multicellular structure with the average length of 160μm and with an average diameter of 28μm,existed in the frond of P.vittata,and the density of trichome on the pinnate axial surface was higher than that on the petiole.Visible X-ray peak of As was recorded in the epidermal cell and trichome.The relative weight of As in the pinnate trichome,which con-tained the highest concentration of As among all tissues of the plant,was 2.4 and 3.9 times as much as that in the epidermal and mesophyllous cells,respectively.The As concentrations in the basal and stalk cells of the same trichome were higher than that in its cap cell.This is the first time to report that the trichome of P.vittata plays an important role in arsenic hyperaccumulation.The finding from the present study implies that much attention should be paid to the role of the trichome in understanding the hyperaccumulation and detoxicity of As in the hyperaccumulator and improving the ability of As accumulation.

Effect of phosphorus on arsenic accumulation in As-hyperaccumulator Pteris vittata L. and its implication

Pot experiment was conducted to understand the effect of phosphorus on arsenic accumulation in As-hyperaccumulator Chinese brake (Pteris vittata L.). It is shown that arsenic concentrations in the fronds and rhizoids, the arsenic bioaccumulation factor, and the total arsenic in the fronds were not influenced significantly under low levels of phosphorus (≤400 mg/kg) and increased sharply under high levels of phosphorus (】400 mg/ kg). The discovery implies that the efficiency of arsenic removal in phytoremedia-tion using the hyperaccumulating plant can be greatly elevated by the phosphorus addition at high rates. The interaction between the accumulation of phosphorus and that of arsenic in plant was stimulated mutually. The result represents that Chinese brake is a good material for plant physiologist to conduct comparative and mechanism studies on the uptake behaviors of phosphorus and arsenic, and phosphorus is also a potential accelerator for phytoremediation of arsenic-contaminated soils.

Distributions of arsenic and essential elements in pinna of arsenic hyperaccumulator Pteris vittata L.

The distributions of arsenic and 6 essential elements in the pinna of As hyperaccumulator, Pteris vittata L., were studied using synchrotron radiation X-ray fluorescence (SRXRF). Significant correlation between the distribution and mobility of the elements revealed that SRXRF study on the elemental distribution was feasible to inspect the transportations of elements in plants. The distribution of As in the pinna showed that As had great abilities to be transported in xylem vessels and from xylem to mesophyll. The distribution of K, one of the most mobile elements in plants, was similar to that of As, whereas the distributions of Fe and Ca with less mobility in plants were almost opposite to that of As in the pinna.

Subcellular distribution and compartmentalization of arsenic in Pteris vittata L.

The subcellular distribution of arsenic (As) in Pteris vittata L., an As-hyperaccumulator, was studied to de- termine As compartmentalization and to explore the mecha- nisms that confer As tolerance. When the plant was grown in a nutrient solution without additional As, most of the accu- mulated As was isolated to the cell wall. However, in plants growing in a nutrient solution containing 0.1 or 0.2 mmol/L As, approximately 78% of the total As accumulated within the pinna. The proportions of As accumulation in the cyto- plasmic supernatant fraction were 78% of that in the pinna and 61% of that in the plant. In either treatment group (0.1 or 0.2 mmol/L As), the fraction containing the lowest level of As was the organelle fraction. These results suggest that As accumulates in the pinna where it is primarily distributed in the cytoplasmic supernatant fraction. The role of As com- partmentalization may be intricately linked with As detoxi- fication in P. vittata L.

Elemental allelopathy by an arsenic hyperaccumulating fern,Pteris vittata L.

Aims the functional advantages of arsenic(As)hyperaccumulation by plants are poorly understood.One proposed benefit,termed ele-mental allelopathy,occurs when hyperaccumulated As is cycled from the plant back into the top layer of soil,allowing As hyperaccu-mulators to gain an advantage over intolerant species by increasing soil As concentrations([As])underneath their canopy.to date,there are no studies that detail the presence of increased soil[As]associ-ated with As hyperaccumulators.In this study,we documented vari-ation in the soil[As]associated with the Chinese brake fern,Pteris vittata L.and also compared the effects of environmentally relevant soil and solution[As]on competitor plant growth.Methods Four populations of P.vittata were identified in central Florida,USA.P.vittata tissue samples and soil samples were collected at the base of and at 3 m away from ferns in each population(n=36).Five sample locations were randomly selected from each site,and soils from the base and 3 m away from each fern were collected to examine the effects of naturally occurring soil[As]on the germination and growth of a potential competitor plant(Oxalis stricta).Solutions with increasing[As]were also used to examine the threshold for negative effects of[As]on O.stricta growth.[As]were measured using inductively coupled plasma mass spectrometry(ICP-MS).Important Findings Overall,soil[As]from the base of ferns was nearly twice that of soil 3 m away indicating that ferns hyperaccumulate As.However,ferns and their associated soil,contained different[As]depending on their col-lection site,indicating that these populations accumulate and use[As]differently.O.stricta growth decreased and germination was delayed as solution and soil[As]increased.However,the relative distance from the fern that the soil was collected from did not affect growth,which would be expected with elemental allelopathy.Our results show that P.vittata is associated with higher soil[As]and these concentrations are sufficient to inhibit growth of competitors.However,the absence of a strong inhibitory relationship associated with proximity to the fern across all locations suggests that the possible functional advantages of elemental allelopathy may depend on site specific characteristics.

EFFECT OF ACTIVE COMPOUNDS ISOLATED FROM PTERIS SEMIPINNATA L ON DNA TOPOISOMERASES AND TYROSINE PROTEIN KINASE AND EXPRESSION OF C-MYC IN LUNG ADENOCARCINOMA CELLS

Objective: To study the effect of active compound 6F and A from Pteris semipinnata L.(PsL) on the activities of DNA topoisomerase (TOPO) I and II, activities of cytosolic and membrane TPK, and expression of oncogene c-myc in lung adenocarcinoma cells. Methods: The effect of compound 6F and A on activities of cytosolic and membrane TPK was measured by scintillation counting; the effect of compound A on expression of oncogene c-myc was determined by flow cytometry indirect fluorimetry. Results: compound 6F and A could inhibit the activities of TOPO I, and they strongly inhibited the TOPO II in 0.01 mg/L and 10.0 mg/L respectively. Compound A slightly inhibited the activities of membrane TPK, but not the cytosolic one. Compound A could inhibit the expression of oncogene c-myc. Conclusion: Topoisomerases are target of compound 6F and A. Compound A could slightly inhibit the activities of TPK, and showed an inhibitory effect on the expression of oncogene c-myc.

水相循环对乙醇-水体系液化蜈蚣草产油特性和砷富集影响

砷富集植物的生态友好处置是实现植物修复产业化发展的关键环节,对土壤重金属污染治理高质量发展起到了制约作用。该研究以蜈蚣草为原料,采用乙醇-水作为反应共溶剂体系,考察了乙醇占比和水相循环次数对蜈蚣草水热产油及重金属砷分布的影响。研究结果显示:在乙醇浓度60%、水相循环3次的条件下,蜈蚣草液化生物油产率最高可达51.08%,其热值为29.15 MJ/kg。生物油主要由酯(>80%)、酚、醇、酮、烃等组成。随水相循环次数的增加,生物油中的酯类和酚类物质增加,高沸点化合物会减少,生物油产率和能量回收率分别提高了58%和32.23%;经过水相循环液化后,超过80%的重金属砷主要分布在水相,且砷的浓度增加至215.05 mg/kg,该结果可为砷富集植物无害化后处置提供参考。因此,水相循环利用是砷富集植物液化处置制取高产率生物油与砷富集的有效途径,为富集植物生态友好处置提供了新的思路。

半边旗提取物6F抑制HL-6 0细胞蛋白激酶C活性(英文)

目的 探讨半边旗提取物 6F的细胞毒作用及诱导DNA片段化与蛋白激酶C(PKC)信号转导途径的关系 ,检测 6F对PKC活性的影响。方法 受试对象为HL 6 0细胞 ,超速离心法获得的胞液 (可溶部分 )及颗粒 (不可溶部分 ,包括细胞膜系统及胞核 )部分用作PKC活性测定。经 0 .4g·L- 1磷脂酰丝氨酸 ,0 .0 4g·L- 1甘油二油酸酯激动剂作用酶粗提物后 ,用液体闪烁计数仪计数 [γ 32 P]ATP参入外源底物的量以测定PKC活性。MTT法测定HL 6 0细胞的活力 ,二苯胺法测定 6F诱导DNA片段化程度。结果 在所测试的浓度范围内 (0 .5～ 312 μmol·L- 1) ,化合物 6F显著抑制胞液及颗粒部分PKC活性 ,最大抑制率达 88.6 % ,呈浓度依赖关系 (胞液部分r =0 .781,P <0 .0 5 ,颗粒部分r =0 .931,P <0 .0 1)。 6F诱导HL 6 0细胞DNA片段化及对细胞的毒性作用可被具有致癌作用的PKC激活剂肉豆蔻酸酯 (PMA ,浓度为 6 5nmol·L- 1)拮抗 ,抑制率分别是 30 %和 4 4% (P <0 .0 1)。PMA单独用使HL 6 0细胞线粒体将MTT还原为甲月赞的能力增强 14 % (P <0 .0 1) ,即增强细胞活力。结论化合物 6F是PKC的抑制剂。 6F对HL 6

半边旗中二萜类化合物的超临界CO_2萃取及HPLC-MS分析

目的 建立一种高效准确的提取和分析半边旗中二萜类化合物的方法。方法 以超临界 CO2 与改性剂乙醇组成的复合流体萃取半边旗中二萜类化合物 ,采用正交设计法优化萃取条件 ;建立了 HPL C-大气压化学电离源(APCI) - MS联用的分析方法 ,以选择离子监测 (SIM)方式进行含量测定 ,以选择离子峰面积与总离子流峰面积的比值优选改性剂。主要的二萜类化合物 5 F的准分子离子为 [M- H]- 1。采用 [M- H]- 1为选择监测离子 ,以色谱保留时间和选择监测离子流峰面积定量。结果 超临界流体萃取最佳条件 :2 5 MPa、6 0℃、15 %甲醇、流量 3.0 m L /min;分析条件 :Diamonsil ODS色谱柱 (15 0 mm× 4 .6 mm,5 μm) ,流动相为 CH3CN- 2 mm ol/ L NH4 AC(35∶ 6 5 ) ,流速 1.0 m L / m in,进样 5μL ,线性范围为 0 .0 5～ 2 .5μg,检出限达 0 .4 ng。 5 F的加样回收率为 97.8% (n=3)。结论 本法提取效率高、检测灵敏度高、选择性好 ,可应用于半边旗中二萜类抗癌化合物的深入研究和质量标准的制定。

半边旗提取物5F对不同类型NSCLC细胞的增殖抑制作用

目的观察半边旗提取物5F(Psl5F)对不同类型非小细胞肺癌(NSCLC)细胞株的增殖抑制作用,并找出对其敏感的细胞株类型。方法采用MTT法观察不同浓度Psl5F分别在作用24、48、72 h时对不同类型的NSCLC细胞株(A549、SPCA-I、NCI-H460、PGCL3)生长增殖的影响。结果不同浓度的Psl5F对4种肺癌细胞作用24、48、72 h后,所有NSCLC细胞株生长增殖均受到不同程度的抑制,其中以对NCI-H460肺癌细胞的杀伤作用最强(P<0.01),对A549、SPCA-I、NCI-H460、PGCL3作用72 h后的半数致死量(IC50)分别是12.12、10.74、3.91、4.94 mg/L。结论Psl5F能有效抑制NSCLC细胞株的体外增殖,且对大细胞肺癌NCI-H460最为敏感。

钙离子在半边旗提取物6F的细胞毒及诱导HL-60细胞DNA片段化中的作用

目的 :研究蕨类植物半边旗提取物 6F对HL - 6 0细胞内游离钙浓度 ([Ca2 + ]i)及Bcl- 2蛋白表达的影响 ,Ca2 + 与 6F细胞毒作用及诱导细胞DNA片段化的关系。方法 :用荧光探针Fura - 2 /AM标记细胞内游离Ca2 + ,在荧光分光光度计上测 [Ca2 + ]i;流式细胞仪检测Bcl- 2的表达 ;噻唑蓝 (MTT)法测定细胞成活率 ;二苯胺法测DNA片段化形成率。结果 :6F作用后 ,HL - 6 0细胞内 [Ca2 + ]i 显著升高 ,呈明显的时间剂量效应关系 ;6F降低Bcl- 2的表达 ;于培养基中加 2mmol/LCa2 + 、或加 1mmol/LEDTA络合细胞外钙 ,或加 4 μmol/L钙通道A2 3 187升高细胞内钙浓度 ,均可增强 6F的细胞毒作用 ,但均不影响 6F诱导细胞DNA片段化程度。加入 2 5 0 μmol/LZn2 + 可显著降低 6F所致DNA片段化率 ,并可增强 6F的细胞毒作用。结论 :化合物 6F可显著升高HL - 6 0细胞 [Ca2 + ]i,推测与 6F降低Bcl- 2的表达有关 ;6F诱导HL - 6 0细胞DNA片段化可能是通过Ca2 + -非依赖性DNA酶的作用所致。

半边旗提取物对HL-60细胞的细胞毒作用及对细胞周期的影响

目的 :研究蕨类植物半边旗提取物对HL 60细胞的体外杀伤作用及对细胞周期的影响。方法 :用噻唑蓝 (MTT)法测定细胞成活率 ;用流式细胞术测定细胞周期时相分布。结果 :半边旗乙醇总提取物PSE及纯化合物 5F、6F及A对HL 60细胞有强烈的杀伤作用 ,具明显的时间和剂量效应 ;PSE、5F、6F及A作用HL 60细胞 2 4h后 ,G0 /G1期细胞比例明显下降 ,S期细胞比例明显升高 ,G2 /M期轻度升高 ( 5F作用组例外 )。结论 :半边旗提取物对HL 60细胞有强烈的杀伤作用 ,明显影响细胞周期时相分布。

半边旗抗肿瘤有效成分6F对HL-60细胞的DNA、RNA及蛋白质生物合成的抑制作用

目的 :从 DNA、RNA及蛋白质生物合成的角度探讨半边旗有效成分 6F抗肿瘤的作用机理。方法 :用台盼蓝拒染法测定细胞成活率 ;[3H] - Td R,[3H] - UTP和 [3H] - Leu参入法分别测定细胞 DNA、RNA及蛋白质生物合成的水平。结果 :6F对 HL - 60细胞生长有强烈的抑制作用 ,且具明显的时间和剂量效应关系。 6F明显抑制 HL- 60细胞 DNA、RNA及蛋白质生物合成 ,呈剂量依赖关系。 6 F对蛋白质合成的抑制作用最强。结论 :6F对 HL- 60细胞生长有强烈的抑制作用 ,抑制细胞 DNA、RNA,特别是蛋白质的生物合成是 6F抗肿瘤作用的可能机制之一。

半边旗抗肿瘤有效成分6F对HL-60细胞c-Myc、Bcl-2及PCNA表达的抑制作用

目的 :研究蕨类植物半边旗提取物 6F对 HL- 60细胞内 c- Myc、Bc1 - 2及增殖细胞核抗原 (PCNA)表达的影响 ,探讨其抗肿瘤作用机制。方法 :异硫氰酸荧光素 (FITC)标记的抗体与细胞反应后在流式细胞仪上检测上述三种蛋白的表达水平。结果 :58～ 2 31 nmo1 / L 6F作用 HL- 60细 1 2 h后 ,c- Myc、Bc1 - 2及 PCNA蛋白表达水平明显下降 ,并呈剂量效应关系。结论 :化合物 6F可下调 HL- 60细胞 c- Myc、Bc1 - 2及 PCNA的表达水平 ,推测这是 6F对 HL - 60细胞具有强烈细胞毒作用并诱导其细胞凋亡的机制之一。

5F-Na注射液对人肝癌MHCC97L细胞毒性及凋亡的影响

目的探讨5F-Na对低转移型人肝癌MHCC97L细胞的抗癌效应。方法不同浓度5F-Na注射液(20、40、80μg·m L^(-1))处理MHCC97L细胞,MTT法检测细胞生长抑制率;Annexin V-EGFP染色及半胱氨酸蛋白酶-3活性检测细胞凋亡;流式细胞术检测细胞周期。结果 5F-Na注射液对MHCC97L细胞增殖抑制作用呈明显的时间、剂量依赖关系;不同浓度5F-Na注射液作用细胞24 h后,细胞凋亡比例较对照组明显升高(P<0.05);并可使细胞阻滞于G_2期。结论 5F-Na注射液抑制MHCC97L增殖与阻滞细胞于G_2期,促进凋亡有关。

PsL5F诱导人未分化甲状腺癌FRO细胞凋亡的机制研究

目的研究半边旗5F(PsL5F)对人未分化甲状腺癌FRO细胞的凋亡诱导及其分子机制。方法用PsL5F处理FRO细胞,MTT法测定其对FRO细胞的生长抑制作用;Annexin V-FITC/PI标记法与流式细胞术联用检测PsL5F对FRO细胞的凋亡诱导;用CM-H2DCFDA和Di OD6(3)标记在流式细胞仪上分析PsL5F对FRO细胞内活性氧(ROS)水平和线粒体膜电位(MMP)的影响;Western blot方法分析Bax、Cyto C、凋亡诱导因子(AIF)及截断PARP的水平变化;Caspase-3活性用Caspase-3比色分析试剂盒测定。结果PsL5F对FRO细胞具有显著的生长抑制作用,且呈现剂量和时间依赖性;PsL5F可诱导FRO细胞凋亡,表现出在100 mg/L浓度下,磷脂酰丝氨酸(PS)外翻细胞的比例呈时间依赖性逐渐增加;用100 mg/L PsL5F处理FRO细胞,在1 h时细胞内ROS水平即明显升高,ROS抑制剂还原型谷胱甘肽(GSH)可抑制PsL5F诱导的细胞内ROS水平升高和细胞凋亡;100mg/L PsL5F处理可使FRO细胞MMP逐渐降低,GSH可抑制PsL5F诱导的MMP降低;同时,线粒体膜蛋白组分中Bax和细胞液蛋白组分中Cyto C、AIF逐渐增加;PsL5F处理使FRO细胞Caspase-3活性逐渐升高及其作用底物PARP断裂。结论PsL5F对人未分化甲状腺癌FRO细胞的生长抑制作用是通过诱导细胞凋亡进行的。其中,细胞内ROS水平升高起到了非常重要的第二信使作用,线粒体是其作用的重要靶点。细胞凋亡是通过Bax转位到线粒体膜、引起MMP降低、Cyto C和AIF释放到细胞液、激活Caspases级联反应而进行的。

半边旗提取物6F对HL-60细胞还原型谷胱甘肽(GSH)水平的影响

目的 :观察半边旗提取物 6F对 HL- 60细胞内还原型谷胱甘肽 (GSH)水平的影响 ,从氧化机制探讨其抗肿瘤作用的机理。方法 :用邻苯二醛 (OPT)荧光分光度法测定细胞内还原型谷胱甘肽。结果 :58至 2 31 nmo1 / L 6F作用细胞 2 0 h及 2 31 nmo1 / L 6F作用细胞 6至2 4 h,HL - 60细胞内 GSH含量均明显降低 ,呈明显的时间剂量效应关系。结论 :化合物 6F可降低 HL- 60细胞内还原型谷胱甘肽水平 ,推测通过细胞内氧化机制杀伤 HL- 60细胞并诱导其细胞凋亡是化合物 6F抗肿瘤作用机制之一。