利用抑制消减杂交技术分离辣椒细胞质雄性不育育性恢复相关EST

 以辣椒(Capsicum annuum L. ) 细胞质雄性不育系23A、121A, 和其相应的近等基因恢复系23C、121C为试验材料, 利用抑制消减杂交( SSH) 技术成功构建了CMS恢复基因诱导表达的消减cDNA文库。结合高密度点阵膜杂交差异筛选, 获得了282个阳性克隆。通过测序, 除去重复序列共得到175个Unique ESTs。在GenBank上进行BLAST分析, 55个EST片段未找到对应的同源序列, 可能代表了新基因;120个EST片段找到了对应的同源序列, 包括103个已知功能基因和17个未知功能基因。按照MIPS功能分类法, 将其分为14个功能组, 涉及代谢、胁迫应答、蛋白活性、转录因子、信号转导等多方面的功能。     

IBA对芍药扦插生根的影响及生根过程中相关酶活性的变化

以芍药‘大富贵’品种为材料, 用1 000、2 000和3 000 mg·L^-1 3个浓度的IBA处理进行扦插对比试验, 研究其生根过程中过氧化物酶( POD) 、多酚氧化酶( PPO) 、吲哚乙酸氧化酶( IAAO) 活性的动态变化。结果表明: IBA处理极显著地提高了插穗的生根率、根数和总根长, 其中以2 000 mg·L ^-1为IBA最佳生根浓度, 将生根周期缩短18 d, 并明显提高了生根质量。在生根过程中, 插穗中POD、PPO活性分别呈现“缓慢上升→下降→急剧上升→下降”和“降→升→降”的变化趋势, 但与对照组相比, 处理组的变化更为剧烈, 峰值出现提前了18 d。IAAO活性在对照和处理中呈现不同的变化趋势: 前者为“升→降→升”, 而后者为“降→降→升”。此外, 高活性的POD、PPO有利于不定根的形成, 低活性的IAAO则促进生根。     

中熟蟠桃新品种‘瑞蟠22号’

 ‘瑞蟠22号’系‘幻想’ ב瑞蟠4号’杂交育成的中熟蟠桃新品种。该品种果实发育期为112 d, 平均单果质量182 g。果实扁平形, 白肉, 硬溶质, 味甜, 粘核。     

Relationship between zinc and zinc transporter expression in human lung cancer cells

AIM: To study the changes of zinc transporter gene expression in A-549 cell line exposed to ZnCl2 and N,N,N’,N’-tetrakis 2-pyridylmethyl ethylenediamine (TPEN).METHODS: Human lung cancer cell line A-549 was exposed to different concentrations of ZnCl2 (0, 50, 100, 150, 200 μmol/L) and TPEN (0, 5, 10, 15, 20 μmol/L), respectively. Twelve hours later, the cell viability was measured by MTT (methyl thiazolyl tetrazolium) and levels of zinc transporter mRNA was detected by RT-PCR. Zinquin was used to estimate the intracellular zinc concentrations.RESULTS: A-549 cell viability rate was significantly decreased when exposed to ZnCl2 at concentrations of 150 and 200 μmol/L, and to TPEN. The intracellular zinc concentration was significantly increased when exposed to ZnCl2 and decreased when exposed to TPEN. Zinc transporter (ZnT-1) mRNA level was increased along with the increase in the concentration of ZnCl2 but decreased when exposed to TPEN. The expressions of ZIP-1 and ZIP-10 (Zrt-and Irt-like protein) were increased along with the increase in the concentration of TPEN but decreased when exposed to ZnCl2.CONCLUSION: ZnT-1 expression is induced by zinc supplement. ZIP-1 and ZIP-10 expressions are induced by zinc deficiency and repressed by zinc supplement.     

Effects of cytokines on renovation of acute renal failure in mouse with bone marrow derived mesenchymal stem cell transplantation

AIM: To observe the effects of cytokines on renovation of acute renal failure (ARF) in mouse with bone marrow derived mesenchymal stem cell (MSC) transplantation. METHODS: ARF animal model was induced in mouse by subcutaneous injection of cisplatin. Mice were randomly assigned into 3 groups: normal control group, ARF group and MSC group. After 24 h cisplatin injection, animals were injected intravenously with MSC in MSC group. Animals were sacrificed at 1 d, 4 d, 7 d, 14 d and 28 d after cisplatin injection. The blood urea nitrogen (BUN) and serum creatinine (Scr) were measured. The renal morphologic changes were scored with Paller’s criterion on hematoxylin and eosin (HE) stained sections. The mRNA and protein expressions of HGF, BMP-7, TNF-α and IL-10 were detected by RT-PCR and immunohistochemistry method. RESULTS: After 4 d of cisplatin injection, the BUN and Scr values in MSC group were significantly lower than those in ARF group (P<0.01). After 7 d and 14 d, the values of BUN and Scr in MSC group were still lower than those in ARF group (P<0.01, P<0.05). The renal morphologic scores of MSC group were also lower than those of ARF group. After 7 d, the expressions of HGF, BMP-7 and IL-10 were higher in MSC group than those in ARF group, the expression of TNF-α in MSC group was lower than that in ARF group. CONCLUSION: MSC promotes the recovery of acute renal failure induced by cisplatin. The mechanism may partly depend on paracrine of growth factor and amelioration of inflammatory.     

Assessment of affecting factors in measuring activating platelets with the method of flow cytometry

AIM:To investigate the affecting factors of detecting platelet activation by flow cytometry (FCM).METHODS:Using decoagulant of natrium citricum, anticoagnlated peripheral venous bloods from 6 healthy donors were labeled with the method of three-colour immunofluorescence assay.Platelet activation markers fibrinogen receptor (Fib-R, PAC-1) and P-selectin (CD62P) were measured.In the same time, the reproducibility of FCM was assessed.RESULTS:The platelet activation markers PAC-1 and CD62P at each time point showed significant difference(P<0.05).The ratio was increased with time extending.The positive ratio of PAC-1 and CD62P immediately measured (within 10 min) was 2.7% and 3.5% less than those at time point of 30 min.The results were measured several times under different activation levels.The coefficient of variation was less than 5%.CONCLUSION:In room temperature and with decoagulant of natrium citricum, if the measurements of PAC-1 and CD62P are finished within 30 min after sampling, good reproducibility should be achieved.     

Asymmetric dimethylarginine protects PC12 cells against glutamate-induced excitotoxic damage

AIM: To investigate the effects of asymmetric dimethylaoyoinine (ADMA) on glutamate-induced PC12 cell damage and its mechanisms. METHODS: PC12 cells were treated with different concentrations of glutamate as an in vitro excitotoxic trauma model. The cell viability was measured by MTT assay. Glutamate cytotoxicity was evaluated by lactate dehydrogenase (LDH) release assay. Intercellular reactive oxygen species (ROS) was detected by dihydrorhodamine123 (DHR) staining and flow cytometric (FCM) analysis. Nitric oxide synthase (NOS) activity and nitric oxide (NO) production were detected by using commercial kits with a spectrophotometer. RESULTS: Glutamate at concentrations of 1 mmol/L to 6 mmol/L dose-dependently decreased PC12 cell viability. Pretreatment 30 min with ADMA prior to administration of glutamate significantly attenuated the inhibition of cell viability, LDH release and ROS accumulation induced by glutamate. Pretreatment with ADMA significantly inhibited the increases in NOS activity and NO production caused by glutamate. CONCLUSION: ADMA obviously protects PC12 cells against glutamate-induced excitotoxicity by inhibiting NOS activity, overproduction of NO and accumulation of intracellular ROS.     

Generation of insulin-producing cells from PDX1 and NKX6.1 gene-modified bone marrow mesenchymal stem cells

AIM: To differentiate bone marrow mesenchymal stem cells (BMSCs) into functional insulin-producing cells to produce sufficient pancreatic islet cells for transplantation. METHODS: Recombinant adenovirus vectors carrying PDX1 and NKX6.1 genes were constructed and the bone marrow mesenchymal stem cells were infected by the recombinant adenovirus combined with several cytokines for differentiation. The expressions of PDX1, NKX6.1 and insulin and C-peptide in the differentiated bone marrow mesenchymal stem cells were detected by RT-PCR and Western blotting. After the differentiated bone marrow mesenchymal stem cells were transplanted into subrenal capsule of diabetic mice, cell morphology of the grafts as well as their secretion of insulin and C-peptide were detected. Besides, regulating capacities of grafts on the blood glucose level of the diabetic mice were also detected. RESULTS: BMSCs induced by recombinant adenovirus (pAdxsi-CMV-PDX1/CMV-NKX6.1) and several cytokines showed positive dithizone staining and the expressions of β-cell related molecule such as insulin and glucose transporter 2 were detected by RT-PCR, which showed a sustaining and stable expression. The similar results were showed by Western blotting, immunohistochemical staining and indirect immunofluorescence. The insulin secretions in the cells stimulated with glucose at concentrations of 5.5 and 25 mmol/L in the experimental group were (1 240.4±109.3) mU/L and (3 539.8±245.1) mU/L, respectively, and were significantly higher than those in control group. Moreover, transplantation of the cells to STZ mice in treatment group made serum glucose recover to normal level. CONCLUSION: PDX1 and NKX6.1 gene-modified bone marrow mesenchymal stem cells differentiate into insulin-producing cells in vitro. When these cells transplanted into STZ induced diabetic mice, their serum glucose could return to the normal level and they could live well. Thus this is a promising method for diabetes treatment.