不同强度高压电场对A549肺癌细胞肿瘤生物学特性的影响

目的：探讨不同强度高压电场对A549肺癌细胞肿瘤转移生物学特性的影响。方法：选择处于生长周期的A549细胞,共分为7个组进行研究,其中A-F组为实验组,G组为不施加电场的空白对照组,A施加500V/cm强度高压电场,F组施加1750V/cm的高压电场,电压场强间隔为250V/cm。采取粘附实验、侵袭及转移实验,检验A549细胞在不同强度高压电场中,其肿瘤生物学转移特性的改变。结果：①各实验组与对照组、各实验组之间的细胞粘附能力,均存在显著性差异（P〈0.05）;②电场强度≥750V/cm时,各实验组之间、及其与对照组之间的细胞迁移能力,存在显著性差异（P〈0.05）;③电场强度≥1000V/cm时,各组与对照组间的细胞侵袭能力,存在显著性差异（P〈0.05）;④电场强度为1000-1250V/cm的各组与1500-1750V/cm各组间的细胞侵袭能力,存在显著性差异,有统计学意义（P〈0.05）。结论：不同强度的电场抑制A549肺癌细胞的程度不同,随着强度的增加,A549细胞粘附、迁移和侵袭能力的抑制现象依次出现,并随着电场强度的增加其抑制程度也持续增加。     

细胞电穿孔导入DNA的动态特征

本文以Ｅ．ＣｏｌｉＨＢ１０１为模型细胞,研究了不同幅度和时间常数的ＲＣ放电脉冲条件下,触发细胞穿孔,导入长度分别为４２２和１６８０ｎｍ的超螺旋长丝形质粒ｐＵＣ－１８和ｐＣＰ１０,及精胺缩合后直径为８８ｎｍ的复曲面体的质粒ｐＣＰ１０,测定了相应的细胞转化率。实验结果显示了细胞电穿孔导入ＤＮＡ的动态特征。     

Inhibition of nucleolar protein nucleolin by electroporation with anti-nucleolin antibodies results in an increase of the nucleolar size

Electroporation of exponentially growing human larynx epidermoid carcinoma cells (HEp-2) with a serum against nucleolin, one of the most abundant non-histone nuclear proteins, has shown, 24 h after electroporation, a significant increase in the size of the nucleolus of these cells compared with normal HEp-2 cells (non-electroporated) and electroporated HEp-2 cells in the absence of antinucleolin serum (P < 0.01). Image analysis evaluation of the different nucleolar components proved a major contribution of the dense fibrillar component to the total nucleolar size in cells electroporated with anti-nucleolin antibodies, more than that corresponding to the dense fibrillar component in cells from any of the control groups (P < 0.01), indicating that the reported increase in nucleolar size was due to a marked enlargement of the dense fibrillar regions. These results, in agreement with previous biochemical and molecular biology studies, suggest a pivotal role for nucleolin in pre-rRNA processing and constitute morphological evidence supporting this role. Following nucleolin inhibition, impaired pre-rRNA processing might result in an accumulation of this molecular species in the dense fibrillar component of the nucleolus, where pre-rRNA is first present.     

用电穿孔方法研究 H5N1 禽流感病毒DNA 疫苗对动物的免疫保护作用

为了研究 H5N1 DNA 疫苗对小鼠和鸡的保护效率,用 H5N1 禽流感病毒 HA DNA 疫苗免疫 BALB/c 小鼠和 SPF 鸡 . 小鼠和鸡分别经电穿孔和肌肉注射免疫两次,间隔为 3 周 . 二次免疫后,用致死量的同源病毒进行攻毒实验 . 空白对照组在攻毒后全部死亡,而经电穿孔免疫的小鼠和鸡均获得了完全的保护,并能有效地抑制病毒在小鼠肺脏和鸡泄殖腔的繁殖 . 同时,电穿孔免疫的小鼠和鸡均产生了高水平的特异性抗体 . 经电穿孔免疫的小鼠攻毒后 CTL 反应明显加强 . 这些结果表明, HA DNA 疫苗能有效地保护小鼠和鸡对禽流感病毒的感染,同时也表明电穿孔免疫是 DNA 疫苗免疫的有效途径之一 .     

用电击孔法转染人红细胞生成素基因

用电击孔(electroporation)转染方法已成功地使化学合成的红细胞生成素(EPO)基因在猿猴肾细胞(COS-7)中表达。对转染条件、表达质粒(pSVL-EPO)DNA含量及转染后不同时间表达产率的观察和研究表明:电击孔缓冲液及相应电压的选择与表达产率有关,且表达质粒DNA量在50～100μg,转染细胞存活率在50％左右时,表达效果好。在转染后24h,便可在细胞上清液中测到EPO活性,48～72h达高峰,EPO活性可达9.7U/ml。     

链霉菌质粒pSET152电转化稀有放线菌小单孢菌的研究

利用链霉菌(Streptomyces)噬菌体ΦC31所构建的整合型载体pSET152作为供体质粒,分别以小单孢菌(Micromonospora)40027菌株的萌发孢子和新鲜菌丝体作为受体菌,在不同的电场强度下进行电转化实验,结果表明:以小单孢菌40027菌株萌发孢子为受体菌,未获得电转化子;以小单孢菌40027菌株新鲜菌丝体为受体菌,获得了电转化子。电场强度为13kV/cm时可获得最高转化效率。Southern杂交结果表明:质粒pSET152可通过菌丝体电转化法导入小单孢菌40027菌株,并整合到小单孢菌40027菌株的染色体上,暗示链霉菌噬菌体ΦC31的整合酶基因和整合位点在异源宿主小单孢菌40027菌株中仍具有相同的功能。质粒稳定性检测实验表明:质粒pSET152可稳定地存在于小单孢菌40027菌株中。     

不同电转仪的电转参数、质粒用量和拓扑结构对猪胎儿成纤维细胞转染效率的影响

为获得猪胎儿成纤维细胞(porcine fetal fibroblasts, PFFs)最佳的电转染效率,本研究利用荧光激活细胞分选技术(fluorescence activated cell sorting, FACS)辅助优化NEPA 21和Nucleofector? 2b两种电转仪电转染PFFs细胞的参数,比较不同质粒用量和拓扑结构在ECM^? 830、NEPA 21和Nucleofector? 2b中的转染效率。结果显示：NEPA 21电转PFFs的最佳穿孔参数为脉冲电压200 V,脉冲长度3 ms,脉冲间隔50 ms,脉冲次数3次,脉冲电压衰减幅度10%;Nucleofector? 2b在U-023的转染参数下达到最高转染效率。ECM^? 830和Nucleofector? 2b的最适质粒用量都为10 μg,而NEPA 21为8 μg;超螺旋质粒比线性化质粒的转染效率更高,且3种仪器中Nucleofector? 2b转染效果最佳。本研究综合考虑电转仪、电转参数、质粒用量和拓扑结构的影响因素以优化PFFs的电转条件,为高效制备转基因猪及基因编辑猪的研究奠定基础。     

T7 RNA聚合酶基因表达系统在鱼腥藻7120中构建及hG-CSF的表达

外源基因的表达效率低是蓝藻基因工程发展的瓶颈之一,T7RNA聚合酶表达系统实现了大肠杆菌中外源基因的高效表达,蓝藻与大肠杆菌同为革兰氏阴性菌,具有较高的遗传同源性,在蓝藻中构建T7 RNA聚合酶表达系统有可能提高外源基因在蓝藻中的表达效率。为了在鱼腥藻7120中构建T7RNA聚合酶表达系统,采用重叠延伸PCR技术和酶切连接等方法构建能够表达T7 RNA聚合酶的定点整合载体pEASY-T1-F1-TacT7RNAPCmR-F2以及由T7启动子驱动hG-CSF基因表达的穿梭表达载体pRL-T7-hG-CSF;采用电击转化法将定点整合载体导入野生型鱼腥藻中,通过三亲接合的方法将穿梭表达载体转入已定点整合T7RNA聚合酶的转基因鱼腥藻中。利用PCR技术鉴定外源基因在蓝藻中的存在;RT-PCR方法检测外源基因在蓝藻中的转录情况;Westernblotting实验检测外源基因在蓝藻中的蛋白表达情况。结果表明两种载体构建成功,T7RNA聚合酶基因和hG-CSF基因被转入鱼腥藻中,两个基因均在藻中表达,T7 RNA聚合酶表达系统在鱼腥藻中构建成功,与传统蓝藻表达系统相比,文中在鱼腥藻中构建的T7表达系...     

Quantitative Proteomic Analysis of Cell Responses to Electroporation,a Classical Gene Delivery Approach

Electroporation, as an established nonviral technology for breaching cell membrane, has been accepted for the delivery of nucleic acids. Despite satisfactory delivery efficiencies have been achieved on multiple cell kinds by simply exhausting all possible electrical parameters, electroporation is still inefficient, or even invalid, for various kinds of cells. This is largely due to the lack of comprehensive understanding of cell responses to electrical stimulation at biological aspect. Moreover, a systematically investigation of protein variation of electroporated cells is also required for biosafety evaluation before clinically applying electroporation. By employing quantitative proteomic analysis, the biological mechanism of electroporation is explored from the molecular level. The results reveal that electrical stimulations widely influence many biological processes including nucleic acid stabilization, protein synthesis, cytoskeleton dynamic, inflammation, and cell apoptosis. It is found that several antivirus‐related processes appeared in the enrichment results. Moreover, SAMD9, a broad spectrum antiviral and antitumor factor, is dramatically downregulated on easy‐to‐transfect cells while electroporation can not alter SAMD9 expression on hard‐to‐transfect cells, hinting that electroporation, a pure physical treatment, can induce antivirus‐like defensive responses and the altering of SAMD9 can be used to predict the effectiveness of electroporation on transfecting specific kinds of cells.