Agrobacterium-mediated DNA transfer

Summary Agrobacterium tumefaciens naturally transfers DNA into plant cells and is clearly one of the most effective methods of directed DNA transfer presently available. Two kinds of vectors are commonly used. Cointegrative vectors have the foreign genes incorporated directly into the Ti plasmid. Binary vectors carry two plasmids; the main Ti plasmid where most of the T-DNA has been removed, and a second plasmid containing the foreign genes between the usual border sequences. The vir genes on the main plasmid function to mobilize the foreign genes into a plant cell. Most plant transformation methods follow the procedure of cocultivating wounded tissue with vir-gene-induced bacteria. The cocultivation step is followed by transfer to a selective medium containing antibiotics to kill the bacterium and to allow only growth of transformed tissue. Several selectable markers are available that include resistance to antibiotics, herbicides, or drugs. In addition, several scorable markers such as the bacterial glucuronidase, chloramphenicol acetyl transferase, and the Agrobacterium opine genes are used to verify transformation. Southern blotting and inheritance of transferred genes are ultimately used to demonstrate stable transformation.     

Review on porcine endogenous retrovirus detection assays—impact on quality and safety of xenotransplants

Xenotransplantation of porcine organs, tissues, and cells inherits a risk for xenozoonotic infections. Viable tissues and cells intended for transplantation have to be considered as potentially contaminated non‐sterile products. The demands on microbial testing, based on the regulatory requirements, are often challenging due to a restricted shelf life or the complexity of the product itself. In Europe, the regulatory framework for xenogeneic cell therapy is based on the advanced therapy medicinal products (ATMP) regulation (2007), the EMA CHMP Guideline on xenogeneic cell‐based medicinal products (2009), as well as the WHO and Council of Europe recommendations. In the USA, FDA guidance for industry (2003) regulates the use of xenotransplants. To comply with the regulations, validated test methods need to be established that reveal the microbial status of a transplant within its given shelf life, complemented by strictly defined action alert limits and supported by breeding in specific pathogen‐free (SPF) facilities. In this review, we focus on assays for the detection of the porcine endogenous retroviruses PERV‐A/‐B/‐C, which exhibit highly polymorphic proviral loci in pig genomes. PERVs are transmitted vertically and cannot be completely eliminated by breeding or gene knock out technology. PERVs entail a public health concern that will persist even if no evidence of PERV infection of xenotransplant recipients in vivo has been revealed yet. Nevertheless, infectious risks must be minimized by full assessment of pigs as donors by combining different molecular screening assays for sensitive and specific detection as well as a functional analysis of the infectivity of PERV including an adequate monitoring of recipients.     

小细胞肺癌细胞诱发人脑微血管内皮细胞紧密连接的开放

目的小细胞肺癌易发生脑转移,但其机制尚不完全清楚,本文探讨小细胞肺癌细胞对人脑微血管内皮细胞单层细胞间紧密连接开放的作用。方法应用免疫荧光技术观察人脑微血管内皮细胞间紧密连接结构改变;应用Western blot技术分析小细胞肺癌细胞与微血管内皮细胞单层共培养时紧密连接蛋白occludin的可溶性片段和不可溶性片段在内皮细胞紧密连接处的分布情况;应用跨内皮迁移实验及辣根过氧化物酶渗漏实验分析小细胞肺癌细胞与内皮细胞单层共培养后其跨内皮细胞单层的迁移能力和内皮单层渗透性的改变;利用ROCK特异性抑制剂Y27632,分析Rho/ROCK信号通路是否参与小细胞肺癌细胞跨内皮单层迁移。结果小细胞肺癌细胞能够通过人脑微血管内皮细胞单层迁移,在0h到12h之间随培养时间的延长小细胞肺癌细胞的迁移率逐渐增加;共培养8h时人脑微血管内皮细胞紧密连接处紧密连接结构蛋白(ZO-1和occludin)表达减少,通透性明显增加;ROCK特异性抑制剂Y27632能够阻断小细胞肺癌细胞引起的内皮细胞间紧密连接处紧密连接结构蛋白ZO-1和occludin表达减少、内皮单层通透性的增加及小细胞肺癌细胞跨内皮单层迁移。结论小细胞肺癌细胞与人脑微血管内皮细胞单层共培养诱发人脑微血管内皮细胞间紧密连接的开放,利于小细胞肺癌细胞的跨内皮单层迁移。     

高糖对混合培养的血管内皮及平滑肌细胞产生TGF-β1的影响

目的：探讨体外混合培养的牛脑微血管内皮细胞（BCMEC）和平滑肌细胞（BCMSMC）产生转化生长因子β1（aTGF－β1）的条件,观察高浓度葡萄糖对混合培养细胞产生aTGF－β1的影响。方法：将BCMEC和BCMSMC在体外作混合接触培养,与单独培养的BCMEC或BCMSMC作对照,用^3H-TdR掺入法检测培养上清中TGF－β1的生物活性,用点杂交法检测TGF－β1mRNA表达水平的变化。分别用含5．5、15、25mmol/L D-葡萄糖的培养液将BCMEC和BCMSMC混合培养24h,检测其上清中TGF－β1的生物学活性。结果：混合培养的细胞上清中有aTGF－β1产生,而单儿培养的细胞上清中均无。高糖培养的BCMEC和BCMSMC上清中TGF－β1活性较正常水平葡萄糖培养的显增高。点杂交结果显示,单独培养的BCMEC或BCMSMC均有TGF－β1mRNA表达,混合培养后mRNA水平无明显改变。结论：体外混合接触培养的BCMEC和BCMSMC可以激活TGF－β1,但并未增加TGF－β1的mRNA表达,高糖可使混合培养细胞上清中aTGF－β1的含量增加。     

慢性乙肝病毒感染不同免疫状态患者树突状细胞体外刺激后介导的 T淋巴细胞功能影响的研究

目的探讨通过补肾解毒和健脾解毒两种不同药物血浆体外作用慢性乙型肝炎（chronic hepatitis B,CHB）患者树突状细胞（dendritic cells,DCs）后对DCs功能的改善,比较哪种药物血浆更能促进DCs激活T淋巴细胞的功能提高。方法选择门诊CHB患者30例,根据其免疫状态,分为免疫耐受期组15例,免疫清除期组15例, 抽取每例患者外周血60 mL,分离培养出成熟的DCs,在培养第7天加入含补肾解毒（六味甘露饮）与健脾解毒（四君甘露饮）的复方中药血浆促进DCs成熟,另设无药物血浆作为对照。培养至第9天,将DCs负载HBVcore18－27核心肽后与自身的T淋巴细胞共培养72 h,收集T淋巴细胞,应用流式细胞技术检测T细胞表面分子CD3、CD28、CD4、CD8及程序型坏死因子－1（programmed death-1, PD-1）的表达水平。结果与无中药血浆的共培养比较,免疫耐受期组和免疫清除期组的患者经补肾解毒和健脾解毒药物血浆干预后的DCs与T细胞共培养,在一定程度上都能提高CD3、CD4、CD28的表达水平,降低CD8、PD-1的表达水平,差异均有统计学意义（P〈0.05）,且补肾解毒药物血浆对提高免疫耐受组期患者CD28分子表达率、减低T细胞表面PD-1分子的表达率,效果均优于健脾解毒血浆的干预,差异均有统计学意义（P〈0.05）。结论通过补肾解毒、健脾解毒药物血浆对DCs体外干预,并与自身T淋巴细胞共培养的方法,能够促进 CHB患者DCs激活T细胞的功能提高和T细胞表面分子的表达,且补肾解毒法对慢性乙型肝炎病毒（hepatitis B virus, HBV）感染免疫耐受期患者影响更为显著。