表达MDVgB基因重组痘苗病毒的构建及保护性研究

以脂质体转染技术构建了表达鸡马立克氏病毒(MDV)gB基因的重组痘苗病毒(RVV-gB), 表明该病毒在CV-1(猴肾细胞)细胞系内能稳定传代,经腹腔1×106 PFU·羽 -1 将重组病毒、HVT冻干苗及痘苗病毒分别按试验程序,免疫1日龄SPF鸡,并于15日龄以3×10 2PFU·羽-1MDV强毒株GA株攻毒,重组病毒和HVT冻干苗匀获得较高的保护, 结果证明了gB是MDV的宿主保护性抗原.此研究为CTL应答检测奠定了基础.     

全磷和缺磷条件下甘蔗叶悬浮细胞主要生长指标的比较

研究了以甘蔗基因型US66-56-9的心叶为材料，对甘蔗愈伤组织进行诱导和建立甘蔗叶片悬浮细胞系，并对悬浮细胞进行全磷和缺磷处理，然后对二者的生长曲线，细胞含水率及培养基pH等变化进行了比较，结果表明；与全磷条件相比，缺磷胁迫抑制细胞的生长，导致细胞含水率较高，使培养基的pH值在生长过程中基本保持酸性状态。     

植物细胞生长周期中内外源细胞激动素的变化

应用细胞同步培养技术、高压液相色谱分析（ＨＰＬＣ）和酶偶联免疫法（ＥＬＩＳＡ）分析了胡萝卜细胞在悬浮培养时生长分裂周期的变化并对内源激动素类物质（玉米素Ｚ－Ｚｅａｔｉｎ、核糖基玉米素ＺＲ－ｚｅａｔｉｎｒｉｂｏｓｉｄｅ、二氢玉米素ＤＨＺ－ｄｉｈｙｄｒｏｚｅａｔｉｎ、核糖二氢玉米素ＤＨＺＲ－ｄｉｈｙｄｒｏｚｅａｔｉｎｒｉｂｏｓｉｄｅ、异戊烯基核糖基腺嘌呤ＩＰ－ｉｓｏｐｅｎｔｅｎｙｌａｄｅｎｉｎｅ和核糖基异戊烯基腺苷ＩＰＲ－ｉｓｏｐｅｎｔｅｎｙｌａｄｅｎｉｎｅｒｉｂｏｓｉｄｅ）含量进行定量分析．从用荧光显微技术分析ＤＮＡ含量变化结果看出，附加有激动素０．１×１０－６·Ｌ－１的悬浮液中，胡萝卜细胞的分裂周期为８．５ｈ，比不加外源激素的处理短１ｈ．在无外源激素存在的情况下，细胞分裂期中内源细胞分裂素类物质除ＺＲ之外，其余的Ｚ、ＤＨＺ、ＤＨＺＲ、ＩＰ和ＩＰＲ的含量在分裂的第６．５ｈ均有一个峰值出现，ＺＲ在第４．５和８．５ｈ有峰值出现．外加细胞分裂素的处理中．Ｚ、ｉＰ和ｉＰＲ整个周期无明显含量变化，ＤＨＺ和ＤＨＺＲ在第６．５ｈ出现峰值．ＺＲ在第８．５ｈ时也出现峰值．说明附加细胞激动素可协调Ｚ．ｉＰ和ｉＰＲ的积累，加速利用?     

流式细胞术检测TcR Vα24/Vβ11双阳性NKT细胞及其在HCV感染者中的应用

目的研究丙肝病毒感染者外周血NKT细胞含量与HCV感染状态之间的相关性.方法应用流式细胞术检测TcR Vα24/Vβ11双阳性NKT细胞.结果抗-HCV阳性且HCVRNA也阳性的病例NKT细胞数量明显低于抗-HCV阳性而HCV RNA阴性的病例（P〈0.01）,明显低于正常对照组（P〈0.05）.结论由于NKT细胞数量减少或活化受阻,造成HCV的持续感染,不利于病毒的清除;另一方面,由于肝内有HCV的存在,NKT细胞大量向肝内趋化,导致外周血的数量相对减少.     

猴头菌子实体提取物对人肺癌细胞SPCA-1的抑制作用

猴头菌(Hericium erinaceus)子实体的醇提物用不同极性有机溶剂分部提取,得到石油醚、氯仿、乙酸乙酯和正丁醇分部,其中仅石油醚分部能抑制SPCA-1细胞增殖和促进其释放ROS(reactive oxygen species),对石油醚分部的进一步研究发现石油醚分部可诱导SPCA-1细胞早期凋亡和降低G_0/G_1期细胞数量。     

细胞穿透肽介导的小黑麦小孢子遗传转化研究

本研究选用小黑麦小孢子作为供试材料,进行细胞穿透肽介导的植物单倍体细胞遗传转化可行性研究。提取单核靠边期的小黑麦小孢子进行悬浮培养,将细胞穿透肽与报告基因GFP的表达框复合物对小孢子进行转化。转化植株进行了PCR分子检测,并在荧光体视显微镜下观察外源GFP基因的表达情况。共获得21株候选转基因植株,其中染色体自然加倍植株为10株。T1代加倍植株的PCR检测结果显示,加倍的转基因株系中有3个株系的PCR检测结果呈阳性。通过荧光体视显微镜可在转基因植株的幼胚部位观测到GFP蛋白的荧光。以上结果表明外源GFP基因已经整合到小黑麦基因组上并且得到表达并稳定遗传。因此,由细胞穿透肽介导的植物单细胞转化是一种有效、可行的植物单细胞转基因方法。     

Phenethyl isothiocyanate as an anti-nutritional factor attenuates deoxynivalenol-induced IPEC-J2 cell injury through inhibiting ROSmediated autophagy

Deoxynivalenol(DON)is considered to be the most harmful mycotoxin that affects the intestinal health of animals and humans.Phenethyl isothiocyanate(PEITC)in feedstuff is an anti-nutritional factor and impairs nutrient digestion and absorption in the animal intestinal.In the current study,we aimed to explore the effects of PEITC on DON-induced apoptosis,intestinal tight junction disorder,and its potential molecular mechanism in the porcine jejunum epithelial cell line(IPEC-J2).Our results indicated that PEITC treatment markedly alleviated DON-induced cytotoxicity,decreasing the apoptotic cell percentage and pro-apoptotic mRNA/protein levels,and increasing zonula occludens-1(ZO-1),occludin and claudin-1 mRNA/protein expression.Meanwhile,PEITC treatment ameliorated DON-induced an increase of the inducible nitric oxide synthase(iNOS)and cyclooxygenase 2(COX-2)mRNA levels and intracellular reactive oxygen species(ROS)level,and a decrease of glutathione peroxidase 1(GPx1),superoxide dismutase 2(SOD2),catalase(CAT)and heme oxygenase 1(HO-1)mRNA levels.Additionally,PEITC treatment significantly down-regulated autophagy-related protein 5(ATG5),beclin-1 and microtubuleassociated protein 1 light chain 3B(LC3-II)mRNA/protein levels,decreased the number of green fluorescent protein-microtubule-associated protein 1 light-chain 3(GFP-LC3)puncta and phosphatidylinositol 3 kinase(PI3K)protein expression,and up-regulated phospho-protein kinase B(p-Akt)and phospho-mammalian target of rapamycin(p-mTOR)protein expression against DON.However,the activation of autophagy by rapamycin,an autophagy agonist,abolished the protective effects of PEITC against DON-induced cytotoxicity,apoptosis and intestinal tight junction disorder.Collectively,PEITC could confer protection against DON-induced porcine intestinal epithelial cell injury by suppressing ROSmediated autophagy.     

动物细胞培养及微载体技术研究进展

动物细胞培养作为生化工程学科领域中迅速发展起来的生物技术与化学工程结合的新型学科,无论在基础研究和应用研究方面都越来越受到生物技术界的重视,现已成为生化工程学科主要前沿学科之一。文中介绍了动物细胞培养技术,并就微载体细胞培养进行了详细的论述。     

Targeted Knock-in of a Fluorescent Protein Gene into the Chicken Vasa Homolog Locus of Chicken Primordial Germ Cells using CRIS-PITCh Method

In chickens, primordial germ cells (PGCs) are effective targets for advanced genome editing, including gene knock-in. Although a long-term culture system has been established for chicken PGCs, it is necessary to select a gene-editing tool that is efficient and precise for editing the PGC genome while maintaining its ability to contribute to the reproductive system. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and CRISPR-mediated precise integration into the target chromosome (CRIS-PITCh) methods are superior as the donor vector is easier to construct, has high genome editing efficiency, and does not select target cells, compared to the homologous recombination method, which has been conventionally used to generate knock-in chickens. In this study, we engineered knock-in chicken PGCs by integrating a fluorescent protein gene cassette as a fusion protein into the chicken vasa homolog (CVH) locus of chicken PGCs using the CRIS-PITCh method. The knock-in PGCs expressed the fluorescent protein in vitro and in vivo, facilitating the tracking of PGCs. Furthermore, we characterized the efficiency of engineering double knock-in cell lines. Knock-in cell clones were obtained by limiting dilution, and the efficiency of engineering double knock-in cell lines was confirmed by genotyping. We found that 82% of the analyzed clones were successfully knocked-in into both alleles. We suggest that the production of model chicken from the knock-in PGCs can contribute to various studies, such as the elucidation of the fate of germ cells and sex determination in chicken.     

颗粒细胞单层对黄牛孤雌胚胎体外发育的影响

为优化颗粒细胞单层共培养体系,研究了不同类型、不同种属的颗粒细胞单层及不同时间更换培养单层对黄牛孤雌胚胎体外发育的影响.结果表明.就卵裂率、囊胚率和囊胚孵出率而言.壁颗粒细胞单层组与丘颗粒细胞单层组之间无显著差异(P>0.05);来自黄牛、猪和小鼠的颗粒细胞单层在支持黄牛孤雌胚胎体外发育方面无显著差异(P>0.05):就囊胚率而言,共培养的第3天和第6天两次更换单层分别与共培养的第4天更换单层和始终不更换单层的对照组之间无显著差异(P>0.05),但第4天更换单层与对照组之间有显著差异(P<0.05).所以.不同类型和不同种属的颗粒细胞单层都能很好地支持黄牛孤雌胚胎的体外发育.在胚胎的体外发育过程中更换培养单层可以明显提高胚胎的囊胚率和囊胚孵出率,并且在共培养的第4天更换培养单层可以得到较高的囊胚率.