实时荧光定量PCR构建朊病相关细胞因子标准品质粒和标准曲线

为能够用实时荧光定量PCR检测PrP106-126作用的鼠巨噬细胞细胞因子的表达水平,构建目的基因IL-1β、TNF-α、IL-6和内参基因β-actin的标准品质粒和标准曲线。根据GenBank中鼠基因编码区保守序列,设计特异性引物;细胞经PrP106-126作用后,提取总RNA。经反转录、PCR扩增、纯化、连接和转化后,提取质粒并测序鉴定,获得IL-1β、TNFα-、IL-6和β-actin质粒;将质粒梯度稀释,进行荧光定量PCR,获得标准曲线及回归方程。结果显示,产物溶解曲线峰值单一,引物特异性高;标准曲线相关系数r2=0.999,表明线性关系好,成功构建了目的基因和内参基因的标准品质粒和标准曲线。     

假俭草抗寒性与体内碳水化合物、脯氨酸、可溶性蛋白含量的关系

为了阐述假俭草(Eremochloa ophiuroides(Munro.)Hack.)的抗寒性与碳水化合物、脯氨酸、可溶性蛋白含量季节动态变化之间的关系,于2008年10月-2009年5月,研究了在自然温度变化过程中,假俭草E126匍匐茎中碳水化合物、脯氨酸、可溶性蛋白的季节性动态变化及其与半致死温度(LT₅₀)变化的关系。结果表明:在越冬前后假俭草E126匍匐茎LT₅₀呈现先降低后升高的季节变化趋势,可溶性总糖、蔗糖、果糖含量,可溶性总糖淀粉比率以及脯氨酸含量伴随田间气温变化均呈现先升高后降低的趋势,可溶性蛋白、淀粉含量则呈现出先降低又升高的趋势。假俭草E126匍匐茎LT₅₀季节变化与可溶性总糖、蔗糖、果糖含量,可溶性总糖淀粉比率季节变化之间存在极显著负相关(P<0.01),而与淀粉含量、脯氨酸含量、可溶性蛋白含量的季节变化间相关性不显著。因此,可溶性总糖含量、蔗糖含量、果糖含量、可溶性总糖淀粉比率对其冬季抗寒性增强有重要作用。     

MicroRNA-126 enhances radiosensitivity of SGC-7901 cells via targeting inhibition of EZH2

AIM: To investigate the molecular biological mechanisms by which microRNA-126 (miR-126) enhances the radiosensitivity of gastric cancer cells. METHODS: SGC-7901 cells were cultured in vitro. In order to over-express miR-126 in SGC-7901 cells, miR-126 mimic was transfected. The mRNA and protein levels of enhancer of zeste ho-molog 2 (EZH2) were detected by RT-qPCR and Western blot, respectively. The targeting relationship between miR-126 and EZH2 was determined by dual-luciferase reporter assay. To estimate the effect of EZH2 on miR-126-enhanced radiosensitivity of the SGC-7901 cells, the pcDNA3.1-EZH2 vector was also co-transfected with miR-126 mimic, and then CCK-8 assay and flow cytometry were used to detect the viability and apoptotic rate of the cells after radiation. RESULTS: Over-expression of miR-126 significantly inhibited the expression of EZH2 in SGC-7901 cells both at protein and mRNA levels (P<0.05). A direct targeting relationship between miR-126 and EZH2 was confirmed by dual-luciferase reporter assay. Compared with the cells only transfected with miR-126 mimic, co-transfection of pcDNA3.1-EZH2 with miR-126 mimic increased the viability but reduced the apoptosis of the cells treated by radiation (P<0.05). CONCLUSION: Targeting inhibition of EZH2 may be one of the mechanisms by which miR-126 enhances the radiosensitivity of gastric cancer cells.     

miRNA-126 inhibits proliferation,migration and invasion of human lung cancer A549 cells via EGFR/AKT/mTOR pathway

AIM: To investigate the effects of microRNA(miRNA)-126 on the proliferation, migration and invasion of human lung cancer cell lines, and to explore its mechanism. METHODS: The A549 cells were transfected with miRNA-126 agomir by Lipofectamine 2000. The expression of miRNA-126 was detected by real-time PCR. The cell activity was detected by MTT assay. The number of viable A549 cells was counted by the method of Trypan blue exclusion. The cell colony-forming capability was determined by cell colony formation test. The cell migration and invasion abilities were assayed by wound healing and Transwell methods, respectively. The protein levels of p-EGFR, EGFR, p-AKT, AKT, p-mTOR and mTOR were determined by Western blot. RESULTS: The expression level of miRNA-126 was significantly increased in the A549 cells compared with negative control(NC) group and control group(P<0.01). The proliferation of A549 cells was decreased extremely after transfected with the miRNA-126 agomir(P<0.01), so did the result of the cell colony-formation test. The migration and invasion abilities of the lung cancer cells were also significantly inhibited. The protein levels of p-EGFR, p-AKT and p-mTOR were significantly down-regulated compared with NC group and control group(P<0.01). CONCLUSION: Over-expression of miRNA-126 significantly inhibits the proliferation, migration and invasion ability of human lung cancer A549 cells by down-regulation of EGFR/AKT/mTOR pathway.     

斑点叉尾鮰趋化因子SCYA126基因及其可变剪接

通过探针杂交筛选斑点叉尾鮰（Ictalurus punctatus）BAC基因组文库，利用引物步移的方法对阳性克隆BAC047_K12进行序列测定，得到2815bp的SCYA126基因组序列。序列分析表明，SCYA126基因由2个外显子和1个内含子组成；外显子拼接的序列与SCYA126 cDNA序列完全一致，编码92个氨基酸，在N端含有2个相邻的半胱氨酸（CC）和2个不相邻的半胱氨酸，为典型的CC趋化因子亚家族成员；其上游调控序列包含TATA框启动子序列和一些与免疫相关的转录因子结合位点。另外，根据与GenBank接收号为BM029630的EST序列的比较分析，发现SCYA126基因组中的内含子没有被剪接，导致翻译后可能产生N端部分缺失的SCYA126蛋白。在胰脏和肝脏中确认了高表达的包含内含子的mRNA的存在，而且其表达量要明显高于正常剪接的SCYA126mRNA。     

PrP106—126诱导小胶质细胞BV-2抗氧化性能的变化

小胶质细胞活化是朊病的病理学特征之一。朊蛋白多肽PrP106—126具神经毒性,是研究异常腕蛋白（PrPSc）的理想工具。为探讨PrP106—126对小胶质细胞氧化压力的影响。本研究以小胶质细胞BV-2为细胞模型,PrP106—126作用48h,应用MTT和流式细胞仪检测细胞的活化情况,应用分子探针技术对细胞的氧化压力（reactiveoxygenspecies,ROs）进行检测,并通过荧光定量RT—PCR对与R0s相关的酶的mRNA表达进行了测定。结果表明PrPl06—126显著促进小胶质细胞BV-2的活化,并提高胞内的ROS水平;定量RT—PCR显示,PrP106—126显著降低细胞S0D-1（P〈0．01）表达水平、提高胞内Cat（P〈0．01）的表达水平;对Grx、Trx-1、和Trx-2mRNA的表达水平有升高的趋势,但未达到显著水平（P〉0．05）,对SOd-2、GPx、GR无显著性影响（P〉0．05）。从分子水平初步阐明小胶质细胞ROS升高的机理。     

Endocrine stress response and abnormal development in carp (Cyprinus carpio) larvae after exposure of the embryos to PCB 126

To investigate whether PCB 126 exposure duringembryonic development induces an endocrine stressresponse in larval carp, eggs were exposed,containing 0.01% ethanol (vehicle-control), 10^-11,immediately after fertilization, for 48 h to water10^-10 or 10^-9 mol l^-1 PCB in 0.01% ethanol. Eggsincubated in water served as controls. After transferto PCB-free water, mortality, the incidence ofyolk-sac and pe-ricardial oedema, wet and dry weight,rate of skin pigmentation, and whole-body contents ofthe stress hormones ACTH, -MSH and cortisol weredetermined at 48, 96, 144, 168, 192 and 216 hpost-fertilization. Except for the dry weight, allparameters of animals exposed to 10^-10 and 10^-9 moll^-1 PCB increased in a concentration-related manner.However, these changes became evident only at 144 hpost-fertilization, i.e. after resorption of theyolk-sac. Swelling of the yolk sac and pericardiumoccurred, and whole-body ACTH, -MSH and cortisollevels increased. Although animals exposed to 10^-10and 10^-9 mol l^-1 PCB displayed stable but elevatedwhole-body ACTH and -MSH levels until 216 h,whole-body cortisol concentrations gradually decreasedfrom 168 h post-fertilization, and were significantlybelow control values at 216 h post-fertilization.Exposure of the carp embryos to 10^-11 mol l^-1 PCB only increased whole-body -MSH levels. Increased whole-body ACTH and cortisol levels indicate that PCBinduces a stress response in carp larvae, possiblymediated by a disturbed hydromineral balance (oedema).We further suggest that the PCB-stimulated bodypigmentation is mediated by a stimulation of -MSHsecretion.     

斑点叉尾趋化因子SCYA126基因及其可变剪接

通过探针杂交筛选斑点叉尾(Ictalurus punctatus)BAC基因组文库,利用引物步移的方法对阳性克隆BAC047 K12进行序列测定,得到2 815 bp的SCYA126基因组序列。序列分析表明,SCYA126基因由2个外显子和1个内含子组成;外显子拼接的序列与SCYA126cDNA序列完全一致,编码92个氨基酸,在N端含有2个相邻的半胱氨酸(CC)和2个不相邻的半胱氨酸,为典型的CC趋化因子亚家族成员;其上游调控序列包含TATA框启动子序列和一些与免疫相关的转录因子结合位点。另外,根据与GenBank接收号为BM029630的EST序列的比较分析,发现SCYA126基因组中的内含子没有被剪接,导致翻译后可能产生N端部分缺失的SCYA126蛋白。在胰脏和肝脏中确认了高表达的包含内含子的mRNA的存在,而且其表达量要明显高于正常剪接的SCYA126mRNA。[中国水产科学,2007,14(1):1-7]     

1株钩状木霉SX005的鉴定及其复合制剂对黄瓜和辣椒苗生长的影响

通过形态学和分子生物学相结合的方法,明确木霉菌株SX005的分类地位,并以该菌株的分生孢子粉为原料,分别与氨基寡糖素、矿源黄腐酸钾、壳寡糖、海藻精、FUNA-801进行复配,通过盆栽试验测定其复合制剂对黄瓜和辣椒苗生长的影响。结果表明,木霉菌株SX005为钩状木霉菌;其5种木霉复配制剂均能促进黄瓜和辣椒苗地上部分生长,且比单独施用木霉菌效果更好,其中氨基寡糖素与木霉菌复配的制剂对2种作物的地上部分生长促进效果最好,使黄瓜株高提高了172%,辣椒株高提高了59.56%。海藻精或壳寡糖与木霉菌复配的制剂可显著增加黄瓜根质量,氨基寡糖素或矿源黄腐酸钾与木霉菌复配制剂可显著增加辣椒根质量。本研究将为钩状木霉SX005复合制剂应用提供技术支撑。     

miR-126 regulates expression of vascular endothelial growth factor in EA.hy926 cells

AIM：To explore the effects of miR-126 on the expression of vascular endothelial growth factor (VEGF) in vascular endothelial cell line EA.hy926. METHODS：EA.hy926 cells were cultured in vitro and transfected with miR-126 mimics or miR-126 inhibitor by cation-mediated transfection method. The total RNA was extracted from the culture cells 36 h after transfection of miR-126 mimics or miR-126 inhibitor. The expression of VEGF at mRNA and protein levels was detected by real-time PCR and Western blotting. RESULTS：Thirty-six hours after transfection of miR-126 inhibitor at concentration of 50 nmol/L, the expression of VEGF at mRNA and protein levels increased significantly as compared with the negative control (P<001). However, transfection of miR-126 mimics at concentration of 50 nmol/L for 36 h significantly decreased the expression of VEGF at mRNA and protein levels as compared with the negative control. CONCLUSION：miR-126 inhibits the expression of VEGF. VEGF may be one of the target genes regulated by miR-126.