电转染效率影响因素的探讨

目的:探讨影响电转染效率的因素。方法:将CHO细胞和DNA(携带标志基因的质粒)混和后进行电转染,观察电转染前孵育温度、渗透压、电转染参数、细胞周期等对细胞存活率和电转染效率影响。结果:孵育温度不影响细胞的存活率,而影响基因的转染效率;电转染前后低渗条件有利于电转染;延长电转染持续时间和频率而不加大电压有利于电转染;最好选用处于G2/M期的细胞进行电转染。结论:初步认为温度、渗透压、电转染参数、细胞周期等对细胞存活率和电转染效率有一定的影响。     

融合蛋白基因与抗体基因电转染CHO-S细胞的条件摸索优化

旨在对编码融合蛋白和抗体的重组质粒转染CHO-S细胞的转染条件进行摸索优化,提高外源基因的转染效率,从而增加外源蛋白的表达。应用Amaxa Nucleofector-II电转仪,从电转染程序、质粒用量及细胞用量三方面着手,最终发现编码融合蛋白的重组质粒的最佳电转条件为:电转程序U-030,质粒用量20μg/孔,细胞用量1×10~7个/孔;编码抗体的重组质粒的最佳电转条件为:电转程序U-024,质粒用量15μg/孔,细胞用量1×10~7个/孔;实验结果进一步显示抗体重组质粒电转染CHO-S细胞的效果要优于融合蛋白重组质粒,利用高通量细胞筛选仪Clone Pix对转染后重组细胞的阳性克隆数进行统计,证实了该抗体重组质粒的转染效率更高。这为以后的抗体及融合蛋白重组质粒电转染CHO-S细胞提供了一定的数据支持,同时提示不同类型的细胞及外源基因,都需要设计相应的电转染实验进行条件优化,进而为获得高产稳定的生产用细胞株奠定基础。     

脂质体介导法转染肿瘤细胞效率的优化

目的:研究优化影响脂质体转染效率的因素,以提高脂质体转染效率,为相关研究和应用提供参考.方法:以绿色荧光蛋白(GFP)作为报告基因,采用脂质体Lipofectamine 2000包裹pU6H1-GFP-FAK重组质粒转染Caco-2细胞,研究了细胞接种密度、DNA用量、脂质体与DNA的比例、脂质体-DNA复合物的形成时间、细胞与脂质体复合物的孵育时间、血清的有无及细胞的传代次数等因素对脂质体转染效率的影响.结果:2-5次细胞传代,2×105接种密度、4μg DNA用量、2.5:1的脂质体与DNA比例、30min脂质体-DNA复合物形成时间以及6h细胞与复合物孵育时间,转染效率最高.血清在本实验室条件下并不影响转染效率.结论:实验获得的优化条件可以明显提高脂质体对肿瘤细胞的转染效率,可作为有关研究或应用的参考.     

重组腺相关病毒转染神经干细胞球的实验研究

目的:探讨重组腺相关病毒2型(rAAV2)对神经干细胞球的转染能力.方法:①将FITC标记的rAAV2(FITC-rAAV2)分成两组,A组直接与神经干细胞球混合,B组与肝素混匀后再与神经干细胞球混合,孵育30 min后在荧光显微镜下观察;②含有GFP报告基因的rAAV2(rAAV2-GFP)与神经干细胞球孵育30 min后,分成两组:A组继续在培养箱内培养,B组分散成单细胞后移植到大鼠脑内,一个月后分别在荧光显微镜下观察神经干细胞球和大鼠脑组织切片中报告基因的表达情况;③将含有低氧启动子(低氧应答元件,HRE)、VEGF和GFP的rAAV2(rAAV2-HRE-VEGF-GFP)转染神经干细胞球后分为两组:A组在低氧条件下培养,B组在常规条件下培养,72 h后观察报告基因的表达情况.结果:①FITC-rAAV2转染神经干细胞球的结果:A组有明亮的绿色荧光,B组基本无绿色荧光;②rAAV2-GFP转染神经干细胞球后一个月,A、B两组均可以看到绿色荧光;③rAAV2-HRE-VEGF-GFP转染神经干细胞球后72 h,A组可见绿色荧光,B组无绿色荧光.结论:rAAV2可以与神经干细胞球特异性结合,rAAV2携带的外源基因在体内和体外均可以有效表达,rAAV2携带外源基因的表达可以人为调控.     

不同日龄大鼠颈上交感神经节神经元电转染方法

目的 建立一种高效电转染不同日龄大鼠颈上交感神经节(superior cervical sympathetic ganglion,SCG)神经元细胞的方法.提高转染后细胞的成活率、转染效率和干扰效率.方法 用传统的及经改良的神经元培养液分别培养电转染后的7日龄、14日龄和40日龄SD大鼠SCG细胞,24 h后用台盼蓝染色方法观察并计算细胞成活率;通过改变质粒DNA和siRNA与转染液比例,优化转染条件,于转染24h后在共聚焦显微镜下观察并计算转染效率或干扰效率.结果 改良培养液可使14日龄以上SD大鼠SCG细胞转染后成活率达到75％以上,明显高于传统培养液转染后的成活率(P＜0.01),且结果稳定,细胞状态良好,能够满足后续实验研究的要求;优化转染条件后,DNA 的转染率及siRNA的干扰率显著提高,当DNA与转染液比例为1∶100(μg∶μL)时,细胞转染率最高;当siRNA与转染液比例为1∶50(μg∶ μL)时干扰率最高.结论 通过改良神经元培养液及优化转染条件,成功提高了电转染后细胞的成活率、转染效率和干扰效率,利用电转染方法可成功转染不同日龄SD大鼠SCG神经元.     

人源性抗体在HEK293T细胞中瞬时表达条件的优化

以HEK293T细胞为宿主对抗体基因的转染和表达条件进行优化。以GFP(green fluorescence protein)和人源性抗b FGF抗体1A2为报告基因,考察了3种转染试剂磷酸钙,PEI,FuGene HD的转染效率,确定FuGene HD的转染效率最高后,对DNA∶FuGene HD的比例,加入氯喹的浓度,转染后的孵育时间,最佳的低温培养温度以及加入组蛋白抑制剂的浓度进行了优化。结果显示,FuGene HD的转染效率最高,达到56.7%。当转染的DNA=2μg/4×106细胞时,DNA∶FuGene HD的最佳比例为1∶4(W/V);加入氯喹能够增强FuGene HD的转染效率,最佳浓度为100μmol/L,转染后的最佳孵育时间为6h;低温诱导能够提高293T细胞表达抗体的能力,最佳的温度为33℃;加入组蛋白抑制剂丁酸钠或丙戊酸能够明显提高抗体的表达量,丙戊酸的效果优于丁酸钠。通过对转染试剂,转染方法,培养温度,组蛋白抑制剂的加入等条件进行优化使人源性抗bFGF抗体的表达量从1.5mg/L提高到了15.1mg/L。     

弓形虫基因缺陷虫株建立方法的初步研究

目的:应用电穿孔技术转染TgP24基因敲除转染质粒于弓形虫RH,探讨电穿孔技术的应用条件,以及TgP24基因敲除质粒转染虫株在不同哺乳动物细胞中筛选的最适条件.方法:设定所需的电穿孔参数、条件,如电压,电容,脉冲次数,电击杯的大小,电转染缓冲液,电穿孔后,将弓形虫悬浮液分别转移到长有不同贴壁细胞的培养瓶中,37℃,5%CO2(体积分数)的培养箱中培养,观察弓形虫的生长状况,并对不同电穿孔参数、条件下的弓形虫存活率进行比较.12hr后换成加有福来霉素的完全培养基中选择培养,不同时间观察虫体及细胞生长情况;在细胞中选择培养10天后,收集虫体,4℃下福来霉素处理7天,再回复到细胞内用选择培养基培养5天,收集虫体,腹腔注射昆明小鼠,大量收集弓形虫用于提取RNA进行RT-PCR.结果:优化电穿孔条件后的弓形虫存活率得到提高(P＜0.05);在选择浓度为5.0μg/ml-7.5μg/ml的福来霉素培养基中,筛选虫株采用L929细胞做为宿主细胞最为适合;RT-PCR结果显示L929细胞筛选基因敲除虫株的效果较好.结论:初步确定了弓形虫电穿孔技术的应用条件,以及Tg P24基因敲除质粒转染虫株在不同哺乳动物细胞中的最佳筛选条件;获得了较好的转染效率,为进一步研究弓形虫Tg P24基因敲除株的生物学特性打下了良好的基础.     

超声破裂载基因微泡增强心肌细胞报告基因的转染与表达

目的:通过超声破裂载基因微泡介导报告基因心肌细胞转染,探讨其能否增强心肌细胞外源基因转染与表达.方法:以β-galactosidase质粒为报告基因,将其与自制氟碳气体微泡粘附,制备载基因微泡.利用诊断性超声破裂微泡进行体外心肌细胞基因转染;以磷酸钙共沉淀转染为阳性对照并将其以不同方式与超声破裂微泡技术联合应用,以期进一步增强基因转染效果.分别采用原位染色及酶学定量检测β-galactosidase表达水平,同时进行细胞活性检测.结果:超声破裂载基因氟碳气体微泡(PESDA)转染组心肌细胞β-galactosidase表达水平可达单纯质粒转染组60倍(P＜0.01).磷酸钙共沉淀转染3.67倍(P＜0.01)超声强度、微泡浓度对超声破裂介导基因转染效果有明显影响.超声破裂微泡技术与磷酸钙共沉淀联合应用可进一步提高报告基因的表达(P＜0.05),即使在磷酸钙转染后6 h,超声破裂微泡仍能明显增强报告的基因的表达(P＜0 05).结论:超声破裂微泡技术是一种高效基因转染方法,其不但能增加DNA转染,而且增强入胞后基因的表达.超声破裂微泡与其它基因转染技术联合应用能进一步增加基因转染效率.     

人红系分化相关基因高表达提高TF-1细胞系存活能力

目的：检测人红系分化相关基因（EDAG）对TF-1细胞株存活能力的影响。方法：采用电转染法将过表达EDAG的质粒高效转染TF-1细胞株,通过G418筛选得到过表达EDAG的TF-1细胞稳定株,用MTs法检测过表达EDAG的细胞稳定株在撤除细胞因子后的增殖情况,用流式细胞术检测细胞凋亡。结果：用表达GFP的质粒电转染TF-1细胞株,通过综合评价转染效率和细胞状态,确定最佳转染条件为1350V电压电击30ms,电击次数为1次;对得到的细胞稳定株进行检测,其内源EDAG的mRNA和蛋白水平均上调;将细胞稳定株在撤除细胞因子状态下培养,过表达EDAG后细胞存活能力增强、凋亡减少。结论：用电转染方式高效转染TF-1细胞,通过筛选得到过表达EDAG的稳定细胞株,并证实撤除细胞因子后过表达EDAG能提高TF-1细胞系的存活及抗凋亡能力。     

增强型绿色荧光蛋白基因电穿孔转染骨髓间充质干细胞的研究

目的建立大鼠骨髓间充质干细胞的分离、培养方法,探讨电穿孔法介导外源基因转染骨髓间充质干细胞的可行性及转染效率.方法 Ficoll-PaqueTMPlus淋巴细胞分离液分离大鼠骨髓间充质干细胞(rMSCs)并进行原代培养和传代扩增,免疫组化的方法对其初步鉴定.用荧光显微镜、细胞计数法和流式细胞仪分析转染效率.结果电穿孔法可较高效转染rMSCs,转染率为(32.8%±3)%.该条件下电转染后的MSCs其生长曲线与转染前的细胞比较无明显变化.结论优化条件的电穿孔法具有较高的介导外源基因表达于rMSCs的效率,且对rMSCs的生物学行为没有明显影响.     

Antibiotics in the treatment of wounds in the victims of the earthquake in the Armenian SSR

Purulent wounds in 88 victims of the Armenian earthquake with the syndrome of long-term squeezing were examined microbiologically and the experience with using antimicrobial drugs for their treatment was analyzed. In all the cases microbial associations were detected, coliform and nonfermenting gram-negative bacilli, enterococci, anaerobes and yeast-like fungi being isolated from 80, 20, 15 and 25 per cent of the patients respectively. 130 bacterial strains were studied in detail. 80 to 100 per cent of the bacterial isolates were resistant to the antibiotics routinely used in surgical practice, i.e. benzylpenicillin, tetracycline, levomycetin, kanamycin, cephalothin and cefazolin and highly sensitive to new antimicrobial drugs belonging to ureidopenicillins (mezlocillin), quinolones (ciprofloxacin) and cephalosporins of the 3rd generation (cefoperazone and ceftazidime). Retrospective estimation of the antibiotic therapy revealed its inadequacy in 55.7 per cent of the patients for the most part because of the isolates resistance. The 19 variants of the antibiotic use prescribed by the physicians mainly included penicillins, aminoglycosides and their combinations (in 67.2 per cent of the cases). The use of the highly efficient drugs of the groups of ureidopenicillins and quinolones was extremely rare which was likely due to lacking of information on the drugs.     

Histogenesis of the taste buds of the vallate papilla in the in the rat in the postnatal stages of development

The developing taste buds of vallate papillae were studied with electron microscope in rats during the first 7 days after birth. Two types of cells--light and dark--are identified in the taste buds of a one day old animal. The apical parts of dark cells are characterized by numerous dark granules. A distinguishing feature of light cells is the presence of synaptic contacts with afferent intragemmal nerves. On the 4th day of development on the top of the apical parts of the cell, a microvillar apparatus is seen to form, which does not yet communicate with the oral cavity. On the 7th day, basal cells appear in the taste buds. Some of these cells are seen mitotically dividing. The differentiated microvillar apparatus now communicates with oral cavity. The structure of the taste buds is getting similar to that in the adults. The structural and functional peculiarities of the developing taste buds are discussed in association with the period of ontogenesis under consideration.     

Changes in the size of the nucleoli of the Purkinje cells in the canine cerebellum in the postresuscitation period

The changes in the size of Purkinje cell (PC) nucleolus in the lateral and medial cerebellum zones were studied in dogs with different degree of neurologic status recovery after clinical death of various etiology and duration. PC always possess one nucleolus in the control and experimental groups. In the case of complete neurologic status recovery of animals the area of PC nucleolus increases in both zones studied, irrespective of the cause of clinical death. In the case of neurologic disorders the increase in PC nucleolus area is clearly expressed only in the medial zone of the cerebellum, being insignificant in the lateral zone. It is suggested that adaptive characteristics of PC are distinct in the two compared zones, which leads to greater PC vulnerability in the lateral zone during deep hypoxia.     

Frequency of the stages in the cycle of the seminiferous epithelium in the rat

This study determined the optimum number of tubules to be counted per testis cross section, and the number of animals per treatment group, when changes in stage frequencies in the cycle of the seminiferous epithelium are criteria for assessing effects of treatment on spermatogenesis. A data base of 9,672 observed and staged tubules was collected from testicular cross sections of 15 Sprague-Dawley rats. A significant variation between animals was found for the frequencies of Stages I, II, IV, VI, VIII, and XIII. Computer simulation was used to randomly select different combinations of animal and tubule numbers from the observed data. Stage frequency means from each simulation experiment were compared statistically to observed mean frequencies. A model that used data from all 14 stages was analyzed. The following conclusions were made: a) a minimum of 200 tubule cross sections/testis is recommended for estimating stage frequencies; b) for a fixed number of tubules scored, the number of animals sampled is more important than the number of tubules per animal in reducing variance; c) to detect a difference of 2 standard deviations from the mean with a 2% error rate and examining 200 tubules/testis, at least 12 animals must be used per group when assessing all 14 stages; d) when individual stages are examined using 10 animals per group, only Stage VII has 80% or greater power of test (alpha = 0.05) to detect a frequency difference; e) pooling stages into 3-4 groups is recommended to improve the power of detecting a treatment difference.     

Self-organization in the determination of the size of the axial structures in the embryogenesis of the clawed toad

Experiments were performed using X. laevis embryos during gastrulation and neurulation (stages 10, 11 1/2, 12 1/2, 13 1/2, 15 and 18). Part of presumptive epidermis and lateral plate mesoderm was removed, and embryos raised until stage 25. The size of axial structures (notochord, somite mesoderm, central nervous system) was determined using serial histological sections and compared with that of control embryos. In experimental embryos, the size of axial structures was decreased. Until a specific stage of development, close correlation was found between the volume of embryonic compartment corresponding to a particular, structure and the volume of presumptive epidermis and lateral plate mesoderm. This stage is individual for each axial organ: middle gastrula (stage 11 1/2) for notochord, late gastrula (stage 12 1/2) for somite mesoderm, and late neurula (stage 18) for central nervous system. This data suggest that differentiation pattern of ecto-mesodermal rudiment is subject to regulation during gastrulation-neurulation, and subdivision of ectoderm and mesoderm into axial and non-axial tissues is a self-organizing process.     

Role of the mediodorsal nucleus of the thalamus in the function of generalization in the cat

In chronic experiments on cats contribution of the thalamic mediodorsal nucleus to the organization of the processes of generalization and abstraction was studied by means of alternation method. Electrolytic ablation of the nucleus retarded the learning and impeded the initial stages of formation of the generalization function. Mechanisms of thalamic dementia are discussed.