肾上腺皮质肿瘤良恶性鉴别方法探究

肾上腺皮质肿瘤（Adrenocortical Tumor,ACT）是一种常见的肿瘤,存在于人体的内分泌系统中,并且是引发肾上腺皮质癌（Adrcnocortical Carcinoma,ACC）进而导致死亡的主要原因.经典的＂Weiss系统＂在诊断明确性上存在种种无法解决的问题,但随着科技水平的发展,人们开始探索新的途径来鉴别ACT的良恶性,于是开始有了影像学和分子生物学技术等特异性的检查方法,从而进一步增强了诊断的明确性.现通过对比的方式,探究肾上腺皮质肿瘤（Adrenocortical Tumor,ACT）影像学检查和分子生物学技术检查的结果,从而能更准确地鉴别其良恶性.     

果实特异启动子调控薄皮甜瓜ACC合成酶cDNA反义表达载体的构建

应用反义RNA技术抑制甜瓜成熟过程中内源乙烯的合成,从而培育耐贮运品种是解决甜瓜延熟保鲜难题的可行新方法。根据GenBank中甜瓜、黄瓜ACC合成酶基因氨基酸保守序列设计引物,从成熟的薄皮甜瓜(齐甜1号)果肉组织中提取总RNA,经RT-PCR扩增得到约0.7kb的ACC合成酶cDNA片段,将其克隆到质粒载体pGEM-TEasy中,测序表明,该基因为777bp,编码258个氨基酸;从番茄(东农706)叶片组织中提取总DNA,经PCR扩增得到约2.2kb的E8基因片段,将其克隆到质粒载体pGEM-TEasy中,测序表明,该基因为2192bp;以pCAM2301为起始植物表达载体,pCAM-GT为中间载体,成功构建了果实特异启动子(E8)调控薄皮甜瓜ACC合成酶cDNA反义表达载体,采用冻融法将其转入根癌农杆菌LBA4404,得到了完整的Ti质粒表达载体系统。     

Effects of ethylene,abscisic acid and auxin on fruit abscission in water dropwort (Oenanthe stolonifera DC.)

Fruit abscission in water dropwort (Oenanthe stolonifera DC.) was investigated by measuring fruit removal force (FRF) affected by fruit ages and plant hormones. FRF decreased rapidly 30 days after anthesis (DAA). Ethylene evolution increased rapidly at 35 DAA and ABA concentration increased gradually after 20 DAA. ACC and ethephon decreased the FRF of fruit explants and the promoting effect of ACC was delayed by silver thiosulphate and cycloheximide, but not by norbornadiene and actinomycin D. ABA enhanced fruit abscission without increasing ethylene evolution. However, the observation that CoCl₂ delayed abscission induced by ABA indicates possible involvement of ethylene in the ABA effect. Treatment with NAA, fenoprop and IAA had no effect on 30 DAA fruit explants, but IAA delayed abscission of 25 DAA explants. This suggests that the effect of auxins may differ with fruit age when used as control agents.     

Effect of Nutrient Restriction and Compensation on Weight,Serum Indexes and Gene Expression of Chest Fat Adipokines in Mongolian Lamb

To study the effect of nutrient restriction and compensation on weight,serum indexes and gene expression of chest fat adipokine in Mongolian lamb,thirty male lambs were randomly divided into control group (20.26 kg± 3.63 kg) and feed restricted group (21.29 kg±3.00 kg).The experiment lasted for 120 days,including 60 days for feed restriction and 60 days for compensatory feed.At the end of each stage,five lambs from each group were slaughtered and chest fat was collected.Serum concentration of triglycerides,non-esterified fatty acids (NEFA),total cholesterol were measured.Quantitative Real-time PCR were used to determine Adiponectin,Visfatin,PPAR-γ and ACC genes expression.The results showed that:①The lamb body weight of restricted group kept maintain level and that of control group showed a increase tendency.During the compensatory period,the average daily gain (ADG) of restricted group was significantly higher than control group (P< 0.05).②During the restriction period,serum triglycerides concentration of lambs in restricted group was significantly decreased at day 30 and 60 (P< 0.05),and NEFA concentration was significantly increased at day 30 (P< 0.05).After 60 days of nutritional compensation,all serum parameters we tested were not significantly different between restrict group and control group (P >0.05).③During the restriction period,PPAR-γ and ACC gene expression in restricted group were significantly lower than that of control group (P< 0.05),while no significant differences were found during the compensation period (P>0.05).Adiponectin and Visfatin genes expression in chest fat was not significantly changed throughout the experiment (P >0.05).We could know that nutritional restriction reduced fatty acid synthesis and adipocyte differentiation ability,adipose tissue was mobilized energy metabolism,and total cholesterol was recoveried growth on the compensation.However,the ACC and PPAR-γ genes in restricted group were significantly decreased with the body weight increased rapidly during the compensatory stage.Adiponectin and Visfatin were not significantly different between restricted and compensatory stages.     

The role of ethylene in abscission of sweet orange promoted by exogenous application of the abscission agent 5-chloro-3-methyl-4-nitro-1H-pyrazole (CMNP)

Mature ‘Valencia’ fruit were treated with 5-chloro-3-methyl-4-nitro-1H-pyrazole (CMNP), and the ethylene inhibitors aminoethoxy vinyl glycine (AVG) and silver thiosulfate (STS), to assess involvement of ethylene in abscission via CMNP. Fruit detachment force (FDF) and ethylene evolution were measured over time. Accumulation of the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) in peel upon CMNP application was assessed and impact of application of ACC on FDF was estimated when applied 1 cm away from the abscission zone (AZ), to determine its potential as a signaling molecule in promoting fruit loosening. CMNP when applied alone reduced FDF by 50%. AVG and STS inhibited fruit loosening considerably at 500 µM and 10 mM, respectively, when applied with 2 mM CMNP. ACC accumulation in peel was highest 2 days after CMNP application, which preceded the maximum decline in FDF which occurred a day later, indicating that ACC serves as a potential signaling molecule that triggers abscission in the AZ. The results of the study support the hypothesis that abscission via CMNP application is at least partly ethylene dependent. However, the decline in FDF by ACC was less than 50% of control which suggested involvement of another pathway in abscission via CMNP in sweet orange.     

Effect of Environmental Stress and Ethylene on the Expression of LeACO1 Gene

The specific ACC oxidase 1 gene fragment of tomato was cloned and prepared for a probe which was used to hybridize with total RNAs that were extracted from the materials treated with ethylene and different environmental stresses, such as drought, water recovery, flood, wound and temperature, respectively. Northern blot analysis indicated that the expression of LeACO1 was restrained by drought, while strongly induced by water recovery and exogenous ethylene. After treated by flood stress, the expression level of I_eACO1 in leaves had no distinctively changes, while its expression level in stems was rapidly increased. Moreover, the expression of LeACO1 in tomato stems could be induced at the temperature of 37 degree. In addition, wounding had no influence on the expression of LeACO1.     

A non-ACC pathway for ethylene biosynthesis in Botrytis cinerea

Premature softening and tissue senescence occur in kiwifruit infected with Botrytis cinerea. While ethylene production is enhanced in infected fruit and B. cinerea produces ethylene on defined media in vitro the source of ethylene in this pathosystem is unclear. Ethylene production by B. cinerea was enhanced when methionine or ∝-keto-methylthiobutyric acid (KMBA) was added to a defined (modified Pratts) medium. Although 1-aminocyclopropane-1-carboxylic acid (ACC) did not stimulate ethylene production, ∝-aminooxyacetic acid (AOA) was inhibitory suggesting a role for a pyridoxal phosphate mediated enzyme reaction down stream from the methionine/KMBA stimulated ethylene biosynthetic pathway. Cobalt chloride (Co²⁺) was inhibitory, but after a 4-d lag period ethylene production from B. cinerea cultures containing methionine and Co²⁺ reached the same level as those without Co²⁺. [U ¹⁴C] methionine was converted to ¹⁴C-ethylene with high efficiency indicating that it is a direct precursor, while [2,3 ¹⁴C]-ACC did not yield radioactively labelled ethylene. These results suggest that the ethylene biosynthetic pathway in B. cinerea does not involve ACC as a precursor and that the enzyme responsible for synthesising ethylene is similar to, but different from, ACC oxidase from higher plants. The ethylene biosynthetic pathway in B. cinerea is yet to be determined.     

大肠杆菌BL21(DE3)中表达重组蛋白的研究

为了研究在大肠杆菌BL2 1(DE3)中重组蛋白的表达特点 ,探讨重组蛋白在大肠杆菌中表达时可溶性蛋白与包涵体出现的特点 ,从表达载体中表达了植物ACC合成酶 .经SDS PAGE电泳与双波长扫描分析 ,确定目的蛋白随表达时间、菌液密度的增加而增加 .但是其最佳表达时间为 2 5h ,菌液密度OD6 0 0 为 0 6 ,IPTG的最佳浓度为 0 6mmol L ,此时目的蛋白含量占全菌蛋白含量之比最高 .植物ACC合成酶在大肠杆菌中表达时以包涵体的形式存在 ,未能检测到可溶性蛋白的表达     

间隙升温处理对芒果内源多胺和ACC含量的影响

紫花芒果在冷害低温中贮藏，对其进行间隙升温处理。结果表明：芒果发唾冷害以后，其内源ＳＰＤ和ＳＰＭ含量显著增加，但内源ＰＵＴ和ＣＡＤ含量的变化不显著；冷害还导致了ＡＣＣ的累积。间隙升温处理显著地减轻了芒果的冷害，延迟了多胺及ＡＣＣ含量的上升。