饲料中添加花粉和酵母硒对中华鳖幼鳖生长和非特异性免疫功能的影响

在基础饲料中分别添加0.5×10-6酵母硒、0.1%花粉、0.5×10-6酵母硒+0.1%花粉的混合物,投喂中华鳖幼鳖90d后。测定幼鳖的生长和血清中谷胱甘肽过氧化物酶(GSH PX)、超氧化物歧化酶(SOD)活性、T淋巴细胞转化率、自然杀伤细胞(NK)活性。同对照组相比,0.5×10-6酵母硒+0.1%花粉组能显著促进中华鳖的生长(P<0.05),0.5×10-6酵母硒组与0.1%花粉组的日增重率均高于对照组,但差异不显著(P>0.05)。各组间成活率无差异(P>0.05)。0.5×10-6酵母硒组NK细胞活性以及血清中SOD酶和GSH PX酶的活力分别比对照组高了156.06%、44.7%、47.5%(P<0.05)。0.1%花粉组、0.5×10-6酵母硒+0.1%花粉组血清中GSH PX酶的活力分别比对照组高了39.9%、32.9%(P<0.05),但两组的SOD酶活力与对照组相比均无显著性差异(P>0.05)。0.5×10-6酵母硒组以及0.1%花粉组的T淋巴细胞转化率均比对照组高,但差异均不显著(P>0.05)。0.1%花粉组的NK细胞活性也比对照组高,但差异不显著(P>0.05)。0.5×10-6酵母硒+0.1%花粉组能显著增强中华鳖T淋巴细胞转化率及NK细胞活性(P<0.05)。     

转基因玉米目标基因纯合体快速准确的PCR鉴定方法(英文)

本文以转基因玉米NK603为例,介绍了一种检测目标基因纯和体的PCR方法.该方法利用4个引物的多重PCR反应,这4个PCR引物分别来自于目标基因的5'端(5'TP)和3'端(3'TP)DNA序列,目标基因5'端一侧的玉米基因组DNA序列(5'GP),以及目标基因3'端一侧的玉米基因组DNA序列(3'GP).视玉米个体有无目标基因以及目标基因的杂合/纯和状态,PCR扩增可得到三种不同的结果:如果被检测个体无目标基因,5'GP与3'GP间的玉米基因组DNA将被扩增,PCR只产生一条条带:如果被检测个体是目标基因的纯合体,5'GP与5'TP及3'TP与3'GP间的DNA将被扩增,PCR则产生两条条带;如果被检测个体是目标基因的杂合体,5'GP与3'GP、5'GP与5'TP以及3'TP与3'GP间的DNA将都被扩增,PCR则产生三条条带.三条不同长短的PCR产物条带在琼脂糖凝胶上清晰可分,易于辨别.和费时昂贵的定量PCR相比,该方法简单、快速、结果准确,在目标基因位点玉米基因组DNA序列已知的前提下,该方法可扩展到诸如Btll、Event176、GA21、MON810、MON863和TC1507等任何转基因玉米的回交转育程序中.     

Perforin expression can define CD8 positive lymphocyte subsets in pigs allowing phenotypic and functional analysis of natural killer, cytotoxic T, natural killer T and MHC un-restricted cytotoxic T-cells

In this study we have used the expression of perforin to characterize subsets of porcine cytotoxic lymphocytes. Perforin positive lymphocytes expressed both CD2 and CD8, most were small dense lymphocytes (SDL) and up to 90% were CD3 negative. However, the numbers of perforin positive T-cells increased with the age of the animal and their populations increased after specific antigen stimulation in vitro. The remaining perforin positive lymphocytes were large and granular and contained more CD3⁺CD5⁺CD6⁺ T-cells (−40%) of which a substantial proportion also co-expressed CD4. Perforin was expressed in subpopulations of both CD8 and CD8β lymphocytes, but was not expressed in γδ T-cells or monocyte/macrophages. The perforin positive CD3⁻ subset was phenotypically homogeneous and defined as CD5⁻CD6⁻CD8β⁻CD16⁺CD11b⁺. This population had NK activity and expressed mRNA for the NK receptor NKG2D, and adaptors DAP10 and DAP12. Perforin positive T-cells (CD3⁺) could be divided into at least three subsets. The first subset was CD4⁻CD5⁺CD6⁺CD11b⁻CD16⁻ most were small dense lymphocytes with cytotoxic T-cell activity but not all expressed CD8β. The second subset was mainly observed in the large granular lymphocytes. Their phenotype was CD4⁺CD5⁺CD6⁺CD8β⁺CD16⁻CD11b⁻ and also showed functional CTL activity. Thus not all of double positive T-cells are memory helper T-cells. The third subset did not express the T-cell co-receptor CD6, but up to half of them expressed another T-cell co-receptor CD5. The majority of this subset expressed CD11b and CD16, thus the third perforin positive T-cell subset was CD3⁺CD4⁻CD5⁺CD6⁻CD8β^±CD11b⁺CD16⁺, and possessed MHC-unrestricted cytotoxicity and LAK activity.     

微量乳酸脱氢酶释放法检测NK细胞活性的几个主要影响因素

对乳酸脱氢酶释放法测定小鼠脾脏 NK细胞活性中的酶促反应时间、效靶细胞比例、检测波长、细胞毒试验孵育时间、维持液中小牛血清 ( FCS)浓度等 5个重要的影响因素进行了探索。结果显示 ,酶促反应时间以 1 0 min最佳 ;效靶细胞比例以 1 5∶ 1为适宜 ;最适检测波长为 570 nm,其结果线性关系好 ;细胞毒试验孵育时间以 2 h为宜 ;维持液中小牛血清以低浓度为宜 ,其结果更接近自然杀伤百分率计算公式 ,并选用 2 %为本试验小牛血清的体积分数     

Biological potentials for a family of disintegrin and metalloproteinase (ADAMDEC)-1 in mouse normal pregnancy

Our previous research has indicated local expression of ADAMDEC-1, a family of disintegrin and metalloproteinase, was confirmed in the mouse placentas and enhancement was found in the sites for spontaneous abortion. Present study was aimed to identify biological effects of ADAMDEC-1 in pregnancy process. Syngeneic pairs of C57BL/6J mice and heterogenic mating pairs of CBA/J and DBA/2 mice were used. Pregnant mice were treated with recombinant ADAMDEC-1 protein. Vasculogenesis effects was evaluated using the Matrigel plugs including vascular endothelial growth factor singularity or combination with ADAMDEC-1. ADAMDEC-1 single effects were evaluated by tubal formation and proliferation assays using HuEht-1 endothelial cells. Expression of ADAMDEC-1 was not exactly corresponded with the time periods for miscarriage initiation. ADAMDEC-1 was distributed in normal placentas and fetuses, especially at extraembryonic ectoderm, decidua cells, uterine natural killer (uNK) cells in decidua, trophoblasts in labyrinthine zone, and hematopoietic cells in umbilical blood and fetal liver. ADAMDEC-1 treatment did not affect reproductive performances, while it elevated uNK cell recruitment in placenta and enlarged lumen sizes of the intraplacental vessels. In vitro analysis also indicated ADAMDEC-1 promoting effect on tubal formation and cell length of HuEht-1. qPCR analysis showed that ADAMDEC-1 modified placental gene expression especially for linkage of actin filament rearrangement. Our findings suggested that ADAMDEC-1 is correlated on cell shape, stability, and movement via modification of actin cytoskeleton. ADMADEC-1 suspected to regulate cellular activity of endothelial cells, trophoblasts, and uNK cells and may support normal developing of mouse placentas.     

转基因玉米NK603基体标准物质研制

转基因安全管理和标识制度的实施需要标准化的检测方法和转基因检测标准物质,转基因标准物质是获得准确、可靠、可比检测结果的保证。目前,转基因玉米NK603已在我国批准进口用作加工原料,其安全监管亟须制备标准物质。本研究将筛选后的转基因玉米NK603杂合种子和对应的非转基因受体种子,按转基因质量分数为60.0mg g^-1、100.0 mg g^-1、1000 mg g^-1 (理论值)的比例配置了3个梯度浓度水平的转基因基体标准物质。利用二重微滴数字聚合酶链式反应(droplet digital polymerase chain reaction, ddPCR)对研制的标准物质进行均匀性与稳定性检测。结果表明:研制的标准物质均匀性良好,可在60℃下运输14 d,稳定性不低于6个月。同时,由8家实验室采用二重ddPCR方法联合测定标准物质NK603转化体与内标基因的拷贝数比值,其标准值及不确定度为:(2.75±0.26)%、(4.68±0.39)%、(51.8±3.7)%。本批标物使用时最小取样量100 mg。可用于转基因食品和饲料...