废电池浸出液对鲫鱼红细胞DNA损伤的研究

应用单细胞凝胶电泳技术(Single cell gel-electrophoresis SCGE)研究了废电池浸出液对鲫鱼红细胞DNA的损伤作用,共分剂量-效应和时间-效应二部分。采用自制废电池浸出液(废电池浸出液为45节1号松下废电池浸泡于40L自来水中90d得到,其中Cu的浓度为28.11μg·L-1,Zn的浓度为3755.61μg·L-1,Pb的浓度为11.70μg·L-1,Cd的浓度为2.01μg·L-1,Hg的浓度为0.77μg·L-1,As的浓度为2.02μg·L-1。),将废电池浸出液与自来水配成5%、10%、20%、30%的溶液进行试验。剂量-效应试验结果表明,5%、10%、20%和30%的废电池浸出液均能在较短时间(48h)内引起鲫鱼红细胞DNA链断裂,出现拖尾现象,即彗星细胞;且随着时间的延长,彗星细胞的数量、彗尾长度和拖尾率均显著增加;DNA损伤程度,在小于2%的范围内,属于中度损伤,在2%～10%的范围内,属于高度损伤,而在大于10%的范围内,属于重度损伤,呈现明显的剂量-效应关系。时间-效应试验结果表明,随着染毒时间的延长,彗星细胞数量增加明显,其中对照组、6h组与其余各组均有显著性差异(P<0.05),彗尾长度及拖尾率也有显著增加,2h内DNA损伤属于中度损伤,2h以后DNA损伤属于高度损伤,呈现明显的时间-效应关系。     

布鲁菌介导细胞自噬的研究进展

布鲁菌是布鲁菌病的病原体,在世界范围内给养殖业带来巨大损失.自噬是细胞的一种代谢方式,细胞在自噬相关基因的调控下清除细胞内病原微生物和吞噬降解受损、衰老细胞器及大分子物质,以维持机体内环境平衡.布鲁菌入侵细胞后,能够诱发细胞自噬,而这一复杂的过程需要很多细胞因子的参与,探究布鲁菌引发的细胞自噬已成为揭示布鲁菌致病机制的...     

The Application and Research Progress of Chicken Embryo and Avian Cell Lines in Biomedicine

Chicken embryo has long been an important experimental model in basic and applied science because of its clear development process,especially in the early development of chicken embryo chorioallantoic membrane stage,due to its abundant blood vessels,it is a natural immunodeficiency host and can be used as an ideal experimental model for pathology,pharmacology and oncology research.The authors briefly described the tissue structure of early stage of chicken embryo,the application of chick embryo chorioallantoic membrane model in tumor,angiogenesis,organ transplantation,burn and other diseases,and the application of anti-cancer drug screening based on the pathological model of chicken embryo.The advances in the application of chicken embryo and avian cell lines in virus reproduction,vaccine production,therapeutic protein production and monoclonal antibody production were reviewed.A variety of human viruses,avian viruses and mycoplasma can proliferate in chicken embryos and avian cell lines and be used in vaccine production.In this paper,the development and characteristics of commonly used avian fibroblasts and pluripotent stem cells were described,and the source of commercial avian cell lines and some susceptible viruses were summarized.Chicken embryo expression system can produce human glycosylates at specific sites of target proteins,reduce the allergic reaction of the target protein to human,and poultry eggs are cheap and easily available,so it can be used as a suitable donor for the production of human monoclonal antibodies and therapeutic proteins.In this paper,the recent progress in the application of chicken embryos and avian cell lines in the field of biomedicine was introduced,and the future application of chicken embryos as animal models was forecasted.     

苜蓿皂苷对大鼠肝脏及肝脏细胞低密度脂蛋白受体、三磷酸腺苷结合盒转运体mRNA表达的影响

本试验旨在研究苜蓿皂苷对大鼠肝脏及大鼠肝脏细胞(BRL细胞)胆固醇清除和转运途径中关键基因低密度脂蛋白受体(LDLR)、三磷酸腺苷结合盒转运体G5(ABCG5)、三磷酸腺苷结合盒转运体G8(ABCG8)mRNA表达量的影响,从个体和细胞水平初步探讨苜蓿皂苷调控胆固醇清除和转运的分子机制。采用高脂饲粮建立大鼠高脂模型,测定苜蓿皂苷对正常、高脂大鼠血清指标[总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)含量]和肝脏LDLR、ABCG5、ABCG8 mRNA表达量的影响;采用高糖DMEM培养液建立BRL细胞脂变模型,测定苜蓿皂苷浓度对BRL细胞活性的影响,测定苜蓿皂苷对正常、脂变细胞LDLR、ABCG5、ABCG8 mRNA表达量的影响。结果表明:1)苜蓿皂苷显著降低高脂大鼠血清中TG、TC和LDL-C的含量(P0.05);2)苜蓿皂苷显著上调正常大鼠肝脏LDLR、ABCG5、ABCG8及高脂大鼠ABCG5、ABCG8 mRNA表达量(P0.05);3)添加200、250μg/m L苜蓿皂苷显著提高了BRL细胞的活性(P0.05);4)苜蓿皂苷显著上调正常BRL细胞LDLR、ABCG5、ABCG8 mRNA表达量(P0.05),而对脂变BRL细胞各基因mRNA表达量无显著影响(P0.05)。结果提示,苜蓿皂苷可通过调控LDLR、ABCG5、ABCG8 mRNA的表达来增加肝细胞内胆固醇的清除和转运,从而降低机体胆固醇的含量。     

稳定表达猪圆环病毒2型Cap蛋白的CHO-K1细胞系的建立及免疫原性分析

建立高表达PCV2-Cap蛋白的CHO-K1悬浮稳转细胞系,鉴定蛋白的免疫原性,为开发新型且有效防治猪圆环病毒的亚单位疫苗奠定基础。构建重组质粒pEE12.4-PCV2-Cap,转染CHO-K1细胞,通过加压筛选,有限稀释,细胞悬浮驯化及Western blot检测得到高表达Cap蛋白的悬浮稳转单克隆细胞株,并对该细胞株进行发酵,纯化得到的目的蛋白,通过小鼠免疫验证其免疫原性。结果表明PCV2-Cap蛋白能够在CHO-K1细胞中正确表达;发酵过程中活细胞密度高达6×10~6个·mL^-1,细胞活力在80%以上,PCV2-Cap蛋白表达量约为370 mg·L^-1;利用间接ELISA检测小鼠免疫后血清中的抗体水平,证明了利用CHO-K1细胞生产的Cap蛋白具备良好的免疫原性。本研究成功构建了表达PCV2-Cap蛋白的CHO-K1悬浮稳转细胞系,并进一步对目的蛋白进行免疫原性分析,为猪圆环病毒亚单位疫苗的开发打下扎实的基础。     

Differentiation and development of Leydig cell, and induction of spermatogenesis during testicular differentiation in the Japanese eel, Anguilla japonica

The initial appearance and the development of Leydig cells (LCs), the sites of steroid hormone production in the testis, were investigated ultrastructurally during testicular differentiation in the Japanese eel, Anguilla japonica. In addition, the effects of a single injection of human chorionic gonadotropin (HCG; 5 IU g body weight^-1) on histological changes of the testes and serum 11-ketotestosterone (11-KT) were examined at various stages (15–18, 20–23, 26–29, 32–35, 38–41 and 46–50 cm body length (BL)) of testicular differentiation. Testicular differentiation was morphologically characterized by the development of loose connective tissue on the medial side in animals 18–29 cm in BL. Ultrastructurally, LCs were first identified in the loose connective tissue of the testis of the 23 cm fish. In the testes of fish over 32 cm, clusters of LCs were distributed throughout the interstitial region accompanying the increase in number of spermatogonia. In fish larger than 32 cm, spermatogenesis was induced by administration of HCG; serum 11-KT levels were also raised. On the other hand, there was no effect on spermatogenesis or serum 11-KT levels in fish less than 29 cm, or in the controls. These result suggests that morphological differentiation of LCs occurs in testis of the 23 cm eel, and subsequently, the testes of eels of BL more than 32 cm acquire the capability to produce steroid hormones.     

草鱼肝细胞脂变模型的建立及脂代谢基因表达分析

为了筛选草鱼肝细胞脂肪变性的最佳诱导剂及浓度,并初步分析脂肪乳剂(lipid emulsions,LE)引起草鱼肝细胞脂肪变性的作用机理,以草鱼(Ctenopharyngodon idellus)正常肝细胞为研究对象,建立草鱼脂肪变性肝细胞模型,以含10%胎牛血清的基础培养液为对照组,处理组为含20%脂肪乳剂0.5~2 m L/L和含20%、50%胎牛血清的诱导培养液,孵育草鱼肝细胞48 h后,定量分析肝细胞内的甘油三酯(TG)含量,观察脂滴积聚情况及肝细胞超微结构的变化,检测细胞培养上清中谷丙转氨酶(alanine transaminase,ALT)、谷草转氨酶(aspartate transaminase,AST)的活性,q RT-PCR技术检测脂代谢关键基因(PPAR?、PPAR?、SREBP-1c、LPL、Lep和UCP2)的转录水平变化,蛋白质印迹技术检测PPAR?、SREBP-1c的蛋白水平变化。结果发现,含1~2 m L/L LE的诱导液组和含20%、50%FBS的诱导液组与对照组相比TG含量均显著上升(P0.05),且20%FBS和各浓度LE诱导组的转氨酶活性与对照组相比差异不显著(P0.05),表明含1~2 m L/L LE的诱导液和含20%FBS的诱导液均可建立草鱼营养性脂肪肝细胞模型。在肝细胞脂变模型中,PPARγ和LPL等脂代谢基因的表达量显著升高(P0.05),而Lep基因表达量显著降低(P0.05),PPARγ和SREBP-1c的蛋白水平升高。结论认为:采用1~2 m L/L LE和20%的FBS均可以在短时间内建立草鱼肝细胞脂肪变性模型,含1 m L/L LE的诱导液诱导效果最佳;肝细胞内脂质的蓄积可能与脂肪代谢关键基因PPARγ、SREBP-1c、LPL及Lep等密切相关。     

生防因子螺旋毛壳ND35的细胞壁降解酶与重寄生作用

1　IntroductionSomespeciesofthesaprophyticascomycetegeneraChaetomiumKunzeexFr .arepotentialantagonistsofseveralplantpathogenicfungi ,forexampleRhizoctoniasolaniandPythiumultimumwhichcauseseedlingdamping -offandVenturiainaequaliswhichcausestheapplescabChae…     

生长激素释放因子在CHO细胞的表达

在对生长激素释放因子 (GRF)基因改造和化学合成 ,并构建了其真核表达载体 pc DNA3- GRF(1- 32 )的基础上 ,用 L ipofectin将上述载体转染 CHO细胞进行瞬时表达。提取转染细胞总 RNA,用 RT- PCR和 Dot blotting检测 GRF基因的表达情况 ,用 Western blotting检测转染细胞上清液的表达产物 ,均得到了阳性结果。制备大鼠垂体单层细胞 ,测定表达产物的生物学活性 ,结果表达产物可刺激生长激素释放 ,并且比对照组提高 3.8倍。试验结果表明 ,已构建的真核表达载体 pc DNA3- GRF(1- 32 )能表达出有生物学活性的 GRF。     

p15基因真核表达重组体及人宫颈癌细胞转染的研究

将含人p15cDNA质粒pBS-SK-p15以EcoRI和XhoI进行双酶切，再与真核表达载体pCR^TM3经相同酶切点连接，转化感受态细菌，筛选，鉴定得到可在真核细胞中表达人p15基因的重组质粒PCR-hp15。以脂质体介导法转染人宫颈癌细胞，用G418筛选，得到转染的人宫颈癌细胞，转染细胞的恶变程度有所改善。